bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2024–08–11
twenty-six papers selected by
Connor Rogerson, University of Cambridge
  1. Nucleosome remodeler exclusion by histone deacetylation enforces heterochromatic silencing and epigenetic inheritance.
  2. Oncogenic transcription factors instruct promoter-enhancer hubs in individual triple negative breast cancer cells.
  3. Lineage-specific canonical and non-canonical activity of EZH2 in advanced prostate cancer subtypes.
  4. FACT mediates the depletion of macroH2A1.2 to expedite gene transcription.
  5. Transcriptional noise, gene activation, and roles of SAGA and Mediator Tail measured using nucleotide recoding single-cell RNA-seq.
  6. Oxidative stress reprograms the transcriptional coactivator Yki to suppress cell proliferation.
  7. The characteristics of CTCF binding sequences contribute to enhancer blocking activity.
  8. DNA polymerase δ subunit Pol32 binds histone H3-H4 and couples nucleosome assembly with Okazaki fragment processing.
  9. DKK1-SE recruits AP1 to activate the target gene DKK1 thereby promoting pancreatic cancer progression.
  10. A DNA condensation code for linker histones.
  11. Stem-loop and circle-loop TADs generated by directional pairing of boundary elements have distinct physical and regulatory properties.
  12. Histone-methyltransferase KMT2D deficiency impairs the Fanconi anemia/BRCA pathway upon glycolytic inhibition in squamous cell carcinoma.
  13. The structure of DNA methyltransferase DNMT3C reveals an activity-tuning mechanism for DNA methylation.
  14. Chromosome structure in Drosophila is determined by boundary pairing not loop extrusion.
  15. Central role of SUMOylation in the regulation of chromatin interactions and transcriptional outputs of the androgen receptor in prostate cancer cells.
  16. An activity-regulated transcriptional program directly drives synaptogenesis.
  17. The DNA methylome of pediatric brain tumors appears shaped by structural variation and predicts survival.
  18. Structural basis of Cdk7 activation by dual T-loop phosphorylation.
  19. Molecular mechanism and functional significance of Wapl interaction with the Cohesin complex.
  20. Single cell dual-omic atlas of the human developing retina.
  21. Blocker-SELEX: a structure-guided strategy for developing inhibitory aptamers disrupting undruggable transcription factor interactions.
  22. SETDB1 suppresses NK cell-mediated immunosurveillance in acute myeloid leukemia with granulo-monocytic differentiation.
  23. Structural dynamics in chromatin unraveling by pioneer transcription factors.
  24. A single-cell transcriptomic map of the developing Atoh1 lineage identifies neural fate decisions and neuronal diversity in the hindbrain.
  25. H3T11 phosphorylation by CKII is required for heterochromatin formation in Neurospora.
  26. Epigenetic reprogramming driving successful and failed repair in acute kidney injury.
  1. Mol Cell. 2024 Jul 26. pii: S1097-2765(24)00582-3. [Epub ahead of print]
    Nucleosome remodeler exclusion by histone deacetylation enforces heterochromatic silencing and epigenetic inheritance.
    Rakesh Kumar Sahu, Jothy Dhakshnamoorthy, Shweta Jain, Hernan Diego Folco, David Wheeler, Shiv I S Grewal.
      Heterochromatin enforces transcriptional gene silencing and can be epigenetically inherited, but the underlying mechanisms remain unclear. Here, we show that histone deacetylation, a conserved feature of heterochromatin domains, blocks SWI/SNF subfamily remodelers involved in chromatin unraveling, thereby stabilizing modified nucleosomes that preserve gene silencing. Histone hyperacetylation, resulting from either the loss of histone deacetylase (HDAC) activity or the direct targeting of a histone acetyltransferase to heterochromatin, permits remodeler access, leading to silencing defects. The requirement for HDAC in heterochromatin silencing can be bypassed by impeding SWI/SNF activity. Highlighting the crucial role of remodelers, merely targeting SWI/SNF to heterochromatin, even in cells with functional HDAC, increases nucleosome turnover, causing defective gene silencing and compromised epigenetic inheritance. This study elucidates a fundamental mechanism whereby histone hypoacetylation, maintained by high HDAC levels in heterochromatic regions, ensures stable gene silencing and epigenetic inheritance, providing insights into genome regulatory mechanisms relevant to human diseases.
    Keywords:  H3K9me3; RSC; SWI/SNF; chromatin remodeling; epigenetic inheritance; gene silencing; heterochromatin; histone deacetylation; histone modifications; read-write
    DOI:  https://doi.org/10.1016/j.molcel.2024.07.006
  2. Sci Adv. 2024 Aug 09. 10(32): eadl4043
    Oncogenic transcription factors instruct promoter-enhancer hubs in individual triple negative breast cancer cells.
    Jingru Zhao, Yeqiao Zhou, Ilias Tzelepis, Noah G Burget, Junwei Shi, Robert B Faryabi.
      Sequencing-based mapping of ensemble pairwise interactions among regulatory elements support the existence of topological assemblies known as promoter-enhancer hubs or cliques in cancer. Yet, prevalence, regulators, and functions of promoter-enhancer hubs in individual cancer cells remain unclear. Here, we systematically integrated functional genomics, transcription factor screening, and optical mapping of promoter-enhancer interactions to identify key promoter-enhancer hubs, examine heterogeneity of their assembly, determine their regulators, and elucidate their role in gene expression control in individual triple negative breast cancer (TNBC) cells. Optical mapping of individual SOX9 and MYC alleles revealed the existence of frequent multiway interactions among promoters and enhancers within spatial hubs. Our single-allele studies further demonstrated that lineage-determining SOX9 and signaling-dependent NOTCH1 transcription factors compact MYC and SOX9 hubs. Together, our findings suggest that promoter-enhancer hubs are dynamic and heterogeneous topological assemblies, which are controlled by oncogenic transcription factors and facilitate subtype-restricted gene expression in cancer.
    DOI:  https://doi.org/10.1126/sciadv.adl4043
  3. Nat Commun. 2024 Aug 08. 15(1): 6779
    Lineage-specific canonical and non-canonical activity of EZH2 in advanced prostate cancer subtypes.
    Varadha Balaji Venkadakrishnan, Adam G Presser, Richa Singh, Matthew A Booker, Nicole A Traphagen, Kenny Weng, Nathaniel C E Voss, Navin R Mahadevan, Kei Mizuno, Loredana Puca, Osasenaga Idahor, Sheng-Yu Ku, Martin K Bakht, Ashir A Borah, Zachary T Herbert, Michael Y Tolstorukov, David A Barbie, David S Rickman, Myles Brown, Himisha Beltran.
      Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase and emerging therapeutic target that is overexpressed in most castration-resistant prostate cancers and implicated as a driver of disease progression and resistance to hormonal therapies. Here we define the lineage-specific action and differential activity of EZH2 in both prostate adenocarcinoma and neuroendocrine prostate cancer (NEPC) subtypes of advanced prostate cancer to better understand the role of EZH2 in modulating differentiation, lineage plasticity, and to identify mediators of response and resistance to EZH2 inhibitor therapy. Mechanistically, EZH2 modulates bivalent genes that results in upregulation of NEPC-associated transcriptional drivers (e.g., ASCL1) and neuronal gene programs in NEPC, and leads to forward differentiation after targeting EZH2 in NEPC. Subtype-specific downstream effects of EZH2 inhibition on cell cycle genes support the potential rationale for co-targeting cyclin/CDK to overcome resistance to EZH2 inhibition.
    DOI:  https://doi.org/10.1038/s41467-024-51156-5
  4. Mol Cell. 2024 Aug 01. pii: S1097-2765(24)00587-2. [Epub ahead of print]
    FACT mediates the depletion of macroH2A1.2 to expedite gene transcription.
    Dengyu Ji, Xue Xiao, Anfeng Luo, Xiongxiong Fan, Jingzhe Ma, Dayi Wang, Miaoran Xia, Lu Ma, Peng-Ye Wang, Wei Li, Ping Chen.
      The histone variant macroH2A is generally linked to transcriptionally inactive chromatin, but how macroH2A regulates chromatin structure and functions in the transcriptional process remains elusive. This study reveals that while the integration of human macroH2A1.2 into nucleosomes does not affect their stability or folding dynamics, it notably hinders the maintenance of facilitates chromatin transcription's (FACT's) function. We show that FACT effectively diminishes the stability of macroH2A1.2-nucleosomes and expedites their depletion subsequent to the initial unfolding process. Furthermore, we identify the residue S139 in macroH2A1.2 as a critical switch to modulate FACT's function in nucleosome maintenance. Genome-wide analyses demonstrate that FACT-mediated depletion of macroH2A-nucleosomes allows the correct localization of macroH2A, while the S139 mutation reshapes macroH2A distribution and influences stimulation-induced transcription and cellular response in macrophages. Our findings provide mechanistic insights into the intricate interplay between macroH2A and FACT at the nucleosome level and elucidate their collective role in transcriptional regulation and immune response of macrophages.
    Keywords:  FACT; histone variant macroH2A; macrophage; nucleosome depletion; transcription
    DOI:  https://doi.org/10.1016/j.molcel.2024.07.011
  5. Cell Rep. 2024 Aug 04. pii: S2211-1247(24)00922-7. [Epub ahead of print]43(8): 114593
    Transcriptional noise, gene activation, and roles of SAGA and Mediator Tail measured using nucleotide recoding single-cell RNA-seq.
    Jeremy A Schofield, Steven Hahn.
      We describe a time-resolved nascent single-cell RNA sequencing (RNA-seq) approach that measures gene-specific transcriptional noise and the fraction of active genes in S. cerevisiae. Most genes are expressed with near-constitutive behavior, while a subset of genes show high mRNA variance suggestive of transcription bursting. Transcriptional noise is highest in the cofactor/coactivator-redundant (CR) gene class (dependent on both SAGA and TFIID) and strongest in TATA-containing CR genes. Using this approach, we also find that histone gene transcription switches from a low-level, low-noise constitutive mode during M and M/G1 to an activated state in S phase that shows both an increase in the fraction of active promoters and a switch to a noisy and bursty transcription mode. Rapid depletion of cofactors SAGA and MED Tail indicates that both factors play an important role in stimulating the fraction of active promoters at CR genes, with a more modest role in transcriptional noise.
    Keywords:  CP: Molecular biology; Mediator; S. cerevisiae; SAGA; bursting; gene activation; single-cell RNA-seq; transcription; transcription noise; yeast
    DOI:  https://doi.org/10.1016/j.celrep.2024.114593
  6. Cell Rep. 2024 Aug 05. pii: S2211-1247(24)00913-6. [Epub ahead of print]43(8): 114584
    Oxidative stress reprograms the transcriptional coactivator Yki to suppress cell proliferation.
    Xiaohan Sun, Dafa Zhou, Yuanfei Sun, Yunhe Zhao, Yanran Deng, Xiaolin Pang, Qingxin Liu, Zizhang Zhou.
      The transcriptional coactivator Yorkie (Yki) regulates organ size by promoting cell proliferation. It is unclear how cells control Yki activity when exposed to harmful stimuli such as oxidative stress. In this study, we show that oxidative stress inhibits the binding of Yki to Scalloped (Sd) but promotes the interaction of Yki with another transcription factor, forkhead box O (Foxo), ultimately leading to a halt in cell proliferation. Mechanistically, Foxo normally exhibits a low binding affinity for Yki, allowing Yki to form a complex with Sd and activate proliferative genes. Under oxidative stress, Usp7 deubiquitinates Foxo to promote its interaction with Yki, thereby activating the expression of proliferation suppressors. Finally, we show that Yki is essential for Drosophila survival under oxidative stress. In summary, these findings suggest that oxidative stress reprograms Yki from a proliferation-promoting factor to a proliferation suppressor, forming a self-protective mechanism.
    Keywords:  CP: Cell biology; CP: Molecular biology; Foxo; Hippo pathway; Usp7; Yki; oxidative stress
    DOI:  https://doi.org/10.1016/j.celrep.2024.114584
  7. Nucleic Acids Res. 2024 Aug 06. pii: gkae666. [Epub ahead of print]
    The characteristics of CTCF binding sequences contribute to enhancer blocking activity.
    Felice H Tsang, Rosa J Stolper, Muhammad Hanifi, Lucy J Cornell, Helena S Francis, Benjamin Davies, Douglas R Higgs, Mira T Kassouf.
      While the elements encoding enhancers and promoters have been relatively well studied, the full spectrum of insulator elements which bind the CCCTC binding factor (CTCF), is relatively poorly characterized. This is partly due to the genomic context of CTCF sites greatly influencing their roles and activity. Here we have developed an experimental system to determine the ability of minimal, consistently sized, individual CTCF elements to interpose between enhancers and promoters and thereby reduce gene expression during differentiation. Importantly, each element is tested in the identical location thereby minimising the effect of genomic context. We found no correlation between the ability of CTCF elements to block enhancer-promoter activity with the degree of evolutionary conservation; their resemblance to the consensus core sequences; or the number of CTCF core motifs harboured in the element. Nevertheless, we have shown that the strongest enhancer-promoter blockers include a previously described bound element lying upstream of the CTCF core motif. In addition, we found other uncharacterised DNaseI footprints located close to the core motif that may affect function. We have developed an assay of CTCF sequences which will enable researchers to sub-classify individual CTCF elements in a uniform and unbiased way.
    DOI:  https://doi.org/10.1093/nar/gkae666
  8. Sci Adv. 2024 Aug 09. 10(32): eado1739
    DNA polymerase δ subunit Pol32 binds histone H3-H4 and couples nucleosome assembly with Okazaki fragment processing.
    Guojun Shi, Chaoqi Yang, Jiale Wu, Yang Lei, Jiazhi Hu, Jianxun Feng, Qing Li.
      During lagging strand chromatin replication, multiple Okazaki fragments (OFs) require processing and nucleosome assembly, but the mechanisms linking these processes remain unclear. Here, using transmission electron microscopy and rapid degradation of DNA ligase Cdc9, we observed flap structures accumulated on lagging strands, controlled by both Pol δ's strand displacement activity and Fen1's nuclease digestion. The distance between neighboring flap structures exhibits a regular pattern, indicative of matured OF length. While fen1Δ or enhanced strand displacement activities by polymerase δ (Pol δ; pol3exo-) minimally affect inter-flap distance, mutants affecting replication-coupled nucleosome assembly, such as cac1Δ and mcm2-3A, do significantly alter it. Deletion of Pol32, a subunit of DNA Pol δ, significantly increases this distance. Mechanistically, Pol32 binds to histone H3-H4 and is critical for nucleosome assembly on the lagging strand. Together, we propose that Pol32 establishes a connection between nucleosome assembly and the processing of OFs on lagging strands.
    DOI:  https://doi.org/10.1126/sciadv.ado1739
  9. Cell Death Dis. 2024 Aug 06. 15(8): 566
    DKK1-SE recruits AP1 to activate the target gene DKK1 thereby promoting pancreatic cancer progression.
    Lan Shao, Haoran Yu, Mengyun Wang, Lu Chen, Boshu Ji, Tong Wu, Xiangqi Teng, Mu Su, Xiao Han, Weikai Shi, Xin Hu, Ziwen Wang, Hongjuan He, Guiping Han, Yan Zhang, Qiong Wu.
      Super-enhancers are a class of DNA cis-regulatory elements that can regulate cell identity, cell fate, stem cell pluripotency, and even tumorigenesis. Increasing evidence shows that epigenetic modifications play an important role in the pathogenesis of various types of cancer. However, the current research is far from enough to reveal the complex mechanism behind it. This study found a super-enhancer enriched with abnormally active histone modifications in pancreatic ductal adenocarcinoma (PDAC), called DKK1-super-enhancer (DKK1-SE). The major active component of DKK1-SE is component enhancer e1. Mechanistically, AP1 induces chromatin remodeling in component enhancer e1 and activates the transcriptional activity of DKK1. Moreover, DKK1 was closely related to the malignant clinical features of PDAC. Deletion or knockdown of DKK1-SE significantly inhibited the proliferation, colony formation, motility, migration, and invasion of PDAC cells in vitro, and these phenomena were partly mitigated upon rescuing DKK1 expression. In vivo, DKK1-SE deficiency not only inhibited tumor proliferation but also reduced the complexity of the tumor microenvironment. This study identifies that DKK1-SE drives DKK1 expression by recruiting AP1 transcription factors, exerting oncogenic effects in PDAC, and enhancing the complexity of the tumor microenvironment.
    DOI:  https://doi.org/10.1038/s41419-024-06915-z
  10. Proc Natl Acad Sci U S A. 2024 Aug 13. 121(33): e2409167121
    A DNA condensation code for linker histones.
    Matthew Watson, Dilyara Sabirova, Megan C Hardy, Yuming Pan, David C J Carpentier, Henry Yates, Charlotte J Wright, W H Chan, Ebru Destan, Katherine Stott.
      Linker histones play an essential role in chromatin packaging by facilitating compaction of the 11-nm fiber of nucleosomal "beads on a string." The result is a heterogeneous condensed state with local properties that range from dynamic, irregular, and liquid-like to stable and regular structures (the 30-nm fiber), which in turn impact chromatin-dependent activities at a fundamental level. The properties of the condensed state depend on the type of linker histone, particularly on the highly disordered C-terminal tail, which is the most variable region of the protein, both between species, and within the various subtypes and cell-type specific variants of a given organism. We have developed an in vitro model system comprising linker histone tail and linker DNA, which although very minimal, displays surprisingly complex behavior, and is sufficient to model the known states of linker histone-condensed chromatin: disordered "fuzzy" complexes ("open" chromatin), dense liquid-like assemblies (dynamic condensates), and higher-order structures (organized 30-nm fibers). A crucial advantage of such a simple model is that it allows the study of the various condensed states by NMR, circular dichroism, and scattering methods. Moreover, it allows capture of the thermodynamics underpinning the transitions between states through calorimetry. We have leveraged this to rationalize the distinct condensing properties of linker histone subtypes and variants across species that are encoded by the amino acid content of their C-terminal tails. Three properties emerge as key to defining the condensed state: charge density, lysine/arginine ratio, and proline-free regions, and we evaluate each separately using a strategic mutagenesis approach.
    Keywords:  chromatin; complex coacervation; intrinsically disordered protein; linker histone; phase separation
    DOI:  https://doi.org/10.1073/pnas.2409167121
  11. Elife. 2024 Aug 07. pii: RP94114. [Epub ahead of print]13
    Stem-loop and circle-loop TADs generated by directional pairing of boundary elements have distinct physical and regulatory properties.
    Wenfan Ke, Miki Fujioka, Paul Schedl, James B Jaynes.
      The chromosomes in multicellular eukaryotes are organized into a series of topologically independent loops called TADs. In flies, TADs are formed by physical interactions between neighboring boundaries. Fly boundaries exhibit distinct partner preferences, and pairing interactions between boundaries are typically orientation-dependent. Pairing can be head-to-tail or head-to-head. The former generates a stem-loop TAD, while the latter gives a circle-loop TAD. The TAD that encompasses the Drosophila even skipped (eve) gene is formed by the head-to-tail pairing of the nhomie and homie boundaries. To explore the relationship between loop topology and the physical and regulatory landscape, we flanked the nhomie boundary region with two attP sites. The attP sites were then used to generate four boundary replacements: λ DNA, nhomie forward (WT orientation), nhomie reverse (opposite of WT orientation), and homie forward (same orientation as WT homie). The nhomie forward replacement restores the WT physical and regulatory landscape: in MicroC experiments, the eve TAD is a 'volcano' triangle topped by a plume, and the eve gene and its regulatory elements are sequestered from interactions with neighbors. The λ DNA replacement lacks boundary function: the endpoint of the 'new' eve TAD on the nhomie side is ill-defined, and eve stripe enhancers activate a nearby gene, eIF3j. While nhomie reverse and homie forward restore the eve TAD, the topology is a circle-loop, and this changes the local physical and regulatory landscape. In MicroC experiments, the eve TAD interacts with its neighbors, and the plume at the top of the eve triangle peak is converted to a pair of 'clouds' of contacts with the next-door TADs. Consistent with the loss of isolation afforded by the stem-loop topology, the eve enhancers weakly activate genes in the neighboring TADs. Conversely, eve function is partially disrupted.
    Keywords:  D. melanogaster; TADs; boundary:boundary pairing; chromatin insulator; chromosome structure; chromosomes; circle-loops; gene expression; pairing orientation; stem-loops
    DOI:  https://doi.org/10.7554/eLife.94114
  12. Nat Commun. 2024 Aug 08. 15(1): 6755
    Histone-methyltransferase KMT2D deficiency impairs the Fanconi anemia/BRCA pathway upon glycolytic inhibition in squamous cell carcinoma.
    Wei Liu, Hongchao Cao, Jing Wang, Areeg Elmusrati, Bing Han, Wei Chen, Ping Zhou, Xiyao Li, Stephen Keysar, Antonio Jimeno, Cun-Yu Wang.
      Histone lysine methyltransferase 2D (KMT2D) is the most frequently mutated epigenetic modifier in head and neck squamous cell carcinoma (HNSCC). However, the role of KMT2D in HNSCC tumorigenesis and whether its mutations confer any therapeutic vulnerabilities remain unknown. Here we show that KMT2D deficiency promotes HNSCC growth through increasing glycolysis. Additionally, KMT2D loss decreases the expression of Fanconi Anemia (FA)/BRCA pathway genes under glycolytic inhibition. Mechanistically, glycolytic inhibition facilitates the occupancy of KMT2D to the promoter/enhancer regions of FA genes. KMT2D loss reprograms the epigenomic landscapes of FA genes by transiting their promoter/enhancer states from active to inactive under glycolytic inhibition. Therefore, combining the glycolysis inhibitor 2-DG with DNA crosslinking agents or poly (ADP-ribose) polymerase (PARP) inhibitors preferentially inhibits tumor growth of KMT2D-deficient mouse HNSCC and patient-derived xenografts (PDXs) harboring KMT2D-inactivating mutations. These findings provide an epigenomic basis for developing targeted therapies for HNSCC patients with KMT2D-inactivating mutations.
    DOI:  https://doi.org/10.1038/s41467-024-50861-5
  13. J Biol Chem. 2024 Aug 02. pii: S0021-9258(24)02134-3. [Epub ahead of print] 107633
    The structure of DNA methyltransferase DNMT3C reveals an activity-tuning mechanism for DNA methylation.
    Nelli Khudaverdyan, Jiuwei Lu, Xinyi Chen, Genevieve Herle, Jikui Song.
      DNA methylation is one of the major epigenetic mechanisms crucial for gene regulation and genome stability. De novo DNA methyltransferase DNMT3C is required for silencing evolutionarily young transposons during mice spermatogenesis. Mutation of DNMT3C led to a sterility phenotype that cannot be rescued by its homologues DNMT3A and DNMT3B. However, the structural basis of DNMT3C-mediated DNA methylation remains unknown. Here, we report the structure and mechanism of DNMT3C-mediated DNA methylation. The DNMT3C methyltransferase domain recognizes CpG-containing DNA in a manner similar to that of DNMT3A and DNMT3B, in line with their high sequence similarity. However, two evolutionary covariation sites, C543 and E590, diversify the substrate interaction among DNMT3C, DNMT3A and DNMT3B, resulting in distinct DNA methylation activity and specificity between DNMT3C, DNMT3A and DNMT3B in vitro. In addition, our combined structural and biochemical analysis reveals that the disease-causing rahu mutation of DNMT3C compromises its oligomerization and DNA-binding activities, providing an explanation for the loss of DNA methylation activity caused by this mutation. This study provides a mechanistic insight into DNMT3C-mediated DNA methylation that complements DNMT3A- and DNMT3B-mediated DNA methylation in mice, unravelling a regulatory mechanism by which evolutionary conservation and diversification fine-tunes the activity of de novo DNA methyltransferases.
    DOI:  https://doi.org/10.1016/j.jbc.2024.107633
  14. Elife. 2024 Aug 07. pii: RP94070. [Epub ahead of print]13
    Chromosome structure in Drosophila is determined by boundary pairing not loop extrusion.
    Xinyang Bing, Wenfan Ke, Miki Fujioka, Amina Kurbidaeva, Sarah Levitt, Mike Levine, Paul Schedl, James B Jaynes.
      Two different models have been proposed to explain how the endpoints of chromatin looped domains ('TADs') in eukaryotic chromosomes are determined. In the first, a cohesin complex extrudes a loop until it encounters a boundary element roadblock, generating a stem-loop. In this model, boundaries are functionally autonomous: they have an intrinsic ability to halt the movement of incoming cohesin complexes that is independent of the properties of neighboring boundaries. In the second, loops are generated by boundary:boundary pairing. In this model, boundaries are functionally non-autonomous, and their ability to form a loop depends upon how well they match with their neighbors. Moreover, unlike the loop-extrusion model, pairing interactions can generate both stem-loops and circle-loops. We have used a combination of MicroC to analyze how TADs are organized, and experimental manipulations of the even skipped TAD boundary, homie, to test the predictions of the 'loop-extrusion' and the 'boundary-pairing' models. Our findings are incompatible with the loop-extrusion model, and instead suggest that the endpoints of TADs in flies are determined by a mechanism in which boundary elements physically pair with their partners, either head-to-head or head-to-tail, with varying degrees of specificity. Although our experiments do not address how partners find each other, the mechanism is unlikely to require loop extrusion.
    Keywords:  D. melanogaster; TADs; boundary elements; boundary:boundary pairing; chromatin insulators; chromosomes; cohesin loop extrusion; gene activation; gene expression; loop topology
    DOI:  https://doi.org/10.7554/eLife.94070
  15. Nucleic Acids Res. 2024 Aug 06. pii: gkae653. [Epub ahead of print]
    Central role of SUMOylation in the regulation of chromatin interactions and transcriptional outputs of the androgen receptor in prostate cancer cells.
    Kaisa-Mari Launonen, Vera Varis, Niina Aaltonen, Einari A Niskanen, Markku Varjosalo, Ville Paakinaho, Jorma J Palvimo.
      The androgen receptor (AR) is pivotal in prostate cancer (PCa) progression and represents a critical therapeutic target. AR-mediated gene regulation involves intricate interactions with nuclear proteins, with many mediating and undergoing post-translational modifications that present alternative therapeutic avenues. Through chromatin proteomics in PCa cells, we identified SUMO ligases together with nuclear receptor coregulators and pioneer transcription factors within the AR's protein network. Intriguingly, this network displayed a significant association with SUMO2/3. To elucidate the influence of SUMOylation on AR chromatin interactions and subsequent gene regulation, we inhibited SUMOylation using ML-792 (SUMOi). While androgens generally facilitated the co-occupancy of SUMO2/3 and AR on chromatin, SUMOi induced divergent effects dependent on the type of AR-binding site (ARB). SUMOi augmented AR's pioneer-like binding on inaccessible chromatin regions abundant in androgen response elements (AREs) and diminished its interaction with accessible chromatin regions sparse in AREs yet rich in pioneer transcription factor motifs. The SUMOi-impacted ARBs divergently influenced AR-regulated genes; those associated with AR-mediated activation played roles in negative regulation of cell proliferation, while those with AR-mediated repression were involved in pattern formation. In conclusion, our findings underscore the pervasive influence of SUMOylation in shaping AR's role in PCa cells, potentially unveiling new therapeutic strategies.
    DOI:  https://doi.org/10.1093/nar/gkae653
  16. Nat Neurosci. 2024 Aug 05.
    An activity-regulated transcriptional program directly drives synaptogenesis.
    Callista Yee, Yutong Xiao, Hongwen Chen, Anay R Reddy, Bing Xu, Taylor N Medwig-Kinney, Wan Zhang, Alan P Boyle, Wendy A Herbst, Yang Kevin Xiang, David Q Matus, Kang Shen.
      Although the molecular composition and architecture of synapses have been widely explored, much less is known about what genetic programs directly activate synaptic gene expression and how they are modulated. Here, using Caenorhabditis elegans dopaminergic neurons, we reveal that EGL-43/MECOM and FOS-1/FOS control an activity-dependent synaptogenesis program. Loss of either factor severely reduces presynaptic protein expression. Both factors bind directly to promoters of synaptic genes and act together with CUT homeobox transcription factors to activate transcription. egl-43 and fos-1 mutually promote each other's expression, and increasing the binding affinity of FOS-1 to the egl-43 locus results in increased presynaptic protein expression and synaptic function. EGL-43 regulates the expression of multiple transcription factors, including activity-regulated factors and developmental factors that define multiple aspects of dopaminergic identity. Together, we describe a robust genetic program underlying activity-regulated synapse formation during development.
    DOI:  https://doi.org/10.1038/s41593-024-01728-x
  17. Nat Commun. 2024 Aug 08. 15(1): 6775
    The DNA methylome of pediatric brain tumors appears shaped by structural variation and predicts survival.
    Fengju Chen, Yiqun Zhang, Lanlan Shen, Chad J Creighton.
      Structural variation heavily influences the molecular landscape of cancer, in part by impacting DNA methylation-mediated transcriptional regulation. Here, using multi-omic datasets involving >2400 pediatric brain and central nervous system tumors of diverse histologies from the Children's Brain Tumor Network, we report hundreds of genes and associated CpG islands (CGIs) for which the nearby presence of somatic structural variant (SV) breakpoints is recurrently associated with altered expression or DNA methylation, respectively, including tumor suppressor genes ATRX and CDKN2A. Altered DNA methylation near enhancers associates with nearby somatic SV breakpoints, including MYC and MYCN. A subset of genes with SV-CGI methylation associations also have expression associations with patient survival, including BCOR, TERT, RCOR2, and PDLIM4. DNA methylation changes in recurrent or progressive tumors compared to the initial tumor within the same patient can predict survival in pediatric and adult cancers. Our comprehensive and pan-histology genomic analyses reveal mechanisms of noncoding alterations impacting cancer genes.
    DOI:  https://doi.org/10.1038/s41467-024-51276-y
  18. Nat Commun. 2024 Aug 03. 15(1): 6597
    Structural basis of Cdk7 activation by dual T-loop phosphorylation.
    Robert Düster, Kanchan Anand, Sophie C Binder, Maximilian Schmitz, Karl Gatterdam, Robert P Fisher, Matthias Geyer.
      Cyclin-dependent kinase 7 (Cdk7) is required in cell-cycle and transcriptional regulation owing to its function as both a CDK-activating kinase (CAK) and part of transcription factor TFIIH. Cdk7 forms active complexes by associating with Cyclin H and Mat1, and is regulated by two phosphorylations in the activation segment (T loop): the canonical activating modification at T170 and another at S164. Here we report the crystal structure of the human Cdk7/Cyclin H/Mat1 complex containing both T-loop phosphorylations. Whereas pT170 coordinates basic residues conserved in other CDKs, pS164 nucleates an arginine network unique to the ternary Cdk7 complex, involving all three subunits. We identify differential dependencies of kinase activity and substrate recognition on the individual phosphorylations. CAK function is unaffected by T-loop phosphorylation, whereas activity towards non-CDK substrates is increased several-fold by T170 phosphorylation. Moreover, dual T-loop phosphorylation stimulates multisite phosphorylation of the RNA polymerase II (RNAPII) carboxy-terminal domain (CTD) and SPT5 carboxy-terminal repeat (CTR) region. In human cells, Cdk7 activation is a two-step process wherein S164 phosphorylation precedes, and may prime, T170 phosphorylation. Thus, dual T-loop phosphorylation can regulate Cdk7 through multiple mechanisms, with pS164 supporting tripartite complex formation and possibly influencing processivity, while pT170 enhances activity towards key transcriptional substrates.
    DOI:  https://doi.org/10.1038/s41467-024-50891-z
  19. Proc Natl Acad Sci U S A. 2024 Aug 13. 121(33): e2405177121
    Molecular mechanism and functional significance of Wapl interaction with the Cohesin complex.
    Xueying Yuan, Lu Yan, Qinfu Chen, Shukai Zhu, Xinyu Zhou, Ling-Hui Zeng, Mingjie Liu, Xiaojing He, Jun Huang, Weiguo Lu, Long Zhang, Haiyan Yan, Fangwei Wang.
      The ring-shaped Cohesin complex, consisting of core subunits Smc1, Smc3, Scc1, and SA2 (or its paralog SA1), topologically entraps two duplicated sister DNA molecules to establish sister chromatid cohesion in S-phase. It remains largely elusive how the Cohesin release factor Wapl binds the Cohesin complex, thereby inducing Cohesin disassociation from mitotic chromosomes to allow proper resolution and separation of sister chromatids. Here, we show that Wapl uses two structural modules containing the FGF motif and the YNARHWN motif, respectively, to simultaneously bind distinct pockets in the extensive composite interface between Scc1 and SA2. Strikingly, only when both docking modules are mutated, Wapl completely loses the ability to bind the Scc1-SA2 interface and release Cohesin, leading to erroneous chromosome segregation in mitosis. Surprisingly, Sororin, which contains a conserved FGF motif and functions as a master antagonist of Wapl in S-phase and G2-phase, does not bind the Scc1-SA2 interface. Moreover, Sgo1, the major protector of Cohesin at mitotic centromeres, can only compete with the FGF motif but not the YNARHWN motif of Wapl for binding Scc1-SA2 interface. Our data uncover the molecular mechanism by which Wapl binds Cohesin to ensure precise chromosome segregation.
    Keywords:  Cohesin; Wapl; chromosome segregation; mitosis; sister chromatid cohesion
    DOI:  https://doi.org/10.1073/pnas.2405177121
  20. Nat Commun. 2024 Aug 09. 15(1): 6792
    Single cell dual-omic atlas of the human developing retina.
    Zhen Zuo, Xuesen Cheng, Salma Ferdous, Jianming Shao, Jin Li, Yourong Bao, Jean Li, Jiaxiong Lu, Antonio Jacobo Lopez, Juliette Wohlschlegel, Aric Prieve, Mervyn G Thomas, Thomas A Reh, Yumei Li, Ala Moshiri, Rui Chen.
      The development of the retina is under tight temporal and spatial control. To gain insights into the molecular basis of this process, we generate a single-nuclei dual-omic atlas of the human developing retina with approximately 220,000 nuclei from 14 human embryos and fetuses aged between 8 and 23-weeks post-conception with matched macular and peripheral tissues. This atlas captures all major cell classes in the retina, along with a large proportion of progenitors and cell-type-specific precursors. Cell trajectory analysis reveals a transition from continuous progression in early progenitors to a hierarchical development during the later stages of cell type specification. Both known and unrecorded candidate transcription factors, along with gene regulatory networks that drive the transitions of various cell fates, are identified. Comparisons between the macular and peripheral retinae indicate a largely consistent yet distinct developmental pattern. This atlas offers unparalleled resolution into the transcriptional and chromatin accessibility landscapes during development, providing an invaluable resource for deeper insights into retinal development and associated diseases.
    DOI:  https://doi.org/10.1038/s41467-024-50853-5
  21. Nat Commun. 2024 Aug 08. 15(1): 6751
    Blocker-SELEX: a structure-guided strategy for developing inhibitory aptamers disrupting undruggable transcription factor interactions.
    Tongqing Li, Xueying Liu, Haifeng Qian, Sheyu Zhang, Yu Hou, Yuchao Zhang, Guoyan Luo, Xun Zhu, Yanxin Tao, Mengyang Fan, Hong Wang, Chulin Sha, Ailan Lin, Jingjing Qin, Kedan Gu, Weichang Chen, Ting Fu, Yajun Wang, Yong Wei, Qin Wu, Weihong Tan.
      Despite the well-established significance of transcription factors (TFs) in pathogenesis, their utilization as pharmacological targets has been limited by the inherent challenges in modulating their protein interactions. The lack of defined small-molecule binding pockets and the nuclear localization of TFs do not favor the use of traditional tools. Aptamers possess large molecular weights, expansive blocking surfaces and efficient cellular internalization, making them compelling tools for modulating TF interactions. Here, we report a structure-guided design strategy called Blocker-SELEX to develop inhibitory aptamers (iAptamers) that selectively block TF interactions. Our approach leads to the discovery of iAptamers that cooperatively disrupt SCAF4/SCAF8-RNAP2 interactions, dysregulating RNAP2-dependent gene expression, which impairs cell proliferation. This approach is further applied to develop iAptamers blocking WDR5-MYC interactions. Overall, our study highlights the potential of iAptamers in disrupting pathogenic TF interactions, implicating their potential utility in studying the biological functions of TF interactions and in nucleic acids drug discovery.
    DOI:  https://doi.org/10.1038/s41467-024-51197-w
  22. Cell Rep. 2024 Jul 30. pii: S2211-1247(24)00865-9. [Epub ahead of print] 114536
    SETDB1 suppresses NK cell-mediated immunosurveillance in acute myeloid leukemia with granulo-monocytic differentiation.
    Yu-Hsuan Chang, Keita Yamamoto, Takeshi Fujino, Teh-Wei Wang, Emi Sugimoto, Wenyu Zhang, Tomohiro Yabushita, Ken Suzaki, E Christine Pietsch, Barbara A Weir, Ramona Crescenzo, Glenn S Cowley, Ricardo Attar, Ulrike Philippar, Mark Wunderlich, Benjamin Mizukawa, Yi Zheng, Yutaka Enomoto, Yoichi Imai, Toshio Kitamura, Susumu Goyama.
      Monocytic acute myeloid leukemia (AML) responds poorly to current treatments, including venetoclax-based therapy. We conducted in vivo and in vitro CRISPR-Cas9 library screenings using a mouse monocytic AML model and identified SETDB1 and its binding partners (ATF7IP and TRIM33) as crucial tumor promoters in vivo. The growth-inhibitory effect of Setdb1 depletion in vivo is dependent mainly on natural killer (NK) cell-mediated cytotoxicity. Mechanistically, SETDB1 depletion upregulates interferon-stimulated genes and NKG2D ligands through the demethylation of histone H3 Lys9 at the enhancer regions, thereby enhancing their immunogenicity to NK cells and intrinsic apoptosis. Importantly, these effects are not observed in non-monocytic leukemia cells. We also identified the expression of myeloid cell nuclear differentiation antigen (MNDA) and its murine counterpart Ifi203 as biomarkers to predict the sensitivity of AML to SETDB1 depletion. Our study highlights the critical and selective role of SETDB1 in AML with granulo-monocytic differentiation and underscores its potential as a therapeutic target for current unmet needs.
    Keywords:  CP: Cancer; CP: Molecular biology; NKG2D ligands; SETDB1; acute myeloid leukemia; anti-leukemic immunity; interferon-stimulated genes; natural killer cells
    DOI:  https://doi.org/10.1016/j.celrep.2024.114536
  23. Biophys Rev. 2024 Jun;16(3): 365-382
    Structural dynamics in chromatin unraveling by pioneer transcription factors.
    Andrea Orsetti, Daphne van Oosten, Roxana-Geanina Vasarhelyi, Theodor-Marian Dănescu, Jan Huertas, Hugo van Ingen, Vlad Cojocaru.
      Pioneer transcription factors are proteins with a dual function. First, they regulate transcription by binding to nucleosome-free DNA regulatory elements. Second, they bind to DNA while wrapped around histone proteins in the chromatin and mediate chromatin opening. The molecular mechanisms that connect the two functions are yet to be discovered. In recent years, pioneer factors received increased attention mainly because of their crucial role in promoting cell fate transitions that could be used for regenerative therapies. For example, the three factors required to induce pluripotency in somatic cells, Oct4, Sox2, and Klf4 were classified as pioneer factors and studied extensively. With this increased attention, several structures of complexes between pioneer factors and chromatin structural units (nucleosomes) have been resolved experimentally. Furthermore, experimental and computational approaches have been designed to study two unresolved, key scientific questions: First, do pioneer factors induce directly local opening of nucleosomes and chromatin fibers upon binding? And second, how do the unstructured tails of the histones impact the structural dynamics involved in such conformational transitions? Here we review the current knowledge about transcription factor-induced nucleosome dynamics and the role of the histone tails in this process. We discuss what is needed to bridge the gap between the static views obtained from the experimental structures and the key structural dynamic events in chromatin opening. Finally, we propose that integrating nuclear magnetic resonance spectroscopy with molecular dynamics simulations is a powerful approach to studying pioneer factor-mediated dynamics of nucleosomes and perhaps small chromatin fibers using native DNA sequences.
    Keywords:  Chromatin; Pioneer transcription factors; Structural dynamics
    DOI:  https://doi.org/10.1007/s12551-024-01205-6
  24. Dev Cell. 2024 Jul 31. pii: S1534-5807(24)00443-X. [Epub ahead of print]
    A single-cell transcriptomic map of the developing Atoh1 lineage identifies neural fate decisions and neuronal diversity in the hindbrain.
    Jessica C Butts, Sih-Rong Wu, Mark A Durham, Ryan S Dhindsa, Jean-Pierre Revelli, M Cecilia Ljungberg, Olivier Saulnier, Madison E McLaren, Michael D Taylor, Huda Y Zoghbi.
      Proneural transcription factors establish molecular cascades to orchestrate neuronal diversity. One such transcription factor, Atonal homolog 1 (Atoh1), gives rise to cerebellar excitatory neurons and over 30 distinct nuclei in the brainstem critical for hearing, breathing, and balance. Although Atoh1 lineage neurons have been qualitatively described, the transcriptional programs that drive their fate decisions and the full extent of their diversity remain unknown. Here, we analyzed single-cell RNA sequencing and ATOH1 DNA binding in Atoh1 lineage neurons of the developing mouse hindbrain. This high-resolution dataset identified markers for specific brainstem nuclei and demonstrated that transcriptionally heterogeneous progenitors require ATOH1 for proper migration. Moreover, we identified a sizable population of proliferating unipolar brush cell progenitors in the mouse Atoh1 lineage, previously described in humans as the origin of one medulloblastoma subtype. Collectively, our data provide insights into the developing mouse hindbrain and markers for functional assessment of understudied neuronal populations.
    Keywords:  Atoh1; CUT&RUN; DNA binding; brainstem; cerebellum; hindbrain; neural fate decisions; single-cell RNA sequencing; spatial transcriptomics
    DOI:  https://doi.org/10.1016/j.devcel.2024.07.007
  25. Nucleic Acids Res. 2024 Aug 06. pii: gkae664. [Epub ahead of print]
    H3T11 phosphorylation by CKII is required for heterochromatin formation in Neurospora.
    Yuan Tian, Chengcheng Zhang, Xiang Tian, Lu Zhang, Tong Yin, Yunkun Dang, Yi Liu, Huiqiang Lou, Qun He.
      Heterochromatin is a key feature of eukaryotic genomes and is crucial for maintaining genomic stability. In fission yeast, heterochromatin nucleation is mainly mediated by DNA-binding proteins or the RNA interference (RNAi) pathway. In the filamentous fungus Neurospora crassa, however, the mechanism that causes the initiation of heterochromatin at the relics of repeat-induced point mutation is unknown and independent of the classical RNAi pathway. Here, we show that casein kinase II (CKII) and its kinase activity are required for heterochromatin formation at the well-defined 5-kb heterochromatin of the 5H-cat-3 region and transcriptional repression of its adjacent cat-3 gene. Similarly, mutation of the histone H3 phosphorylation site T11 also impairs heterochromatin formation at the same locus. The catalytic subunit CKA colocalizes with H3T11 phosphorylation (H3pT11) within the 5H-cat-3 domain and the deletion of cka results in a significant decrease in H3T11 phosphorylation. Furthermore, the loss of kinase activity of CKII results in a significant reduction of H3pT11, H3K9me3 (histone H3 lysine 9 trimethylation) and DNA methylation levels, suggesting that CKII regulates heterochromatin formation by promoting H3T11 phosphorylation. Together, our results establish that histone H3 phosphorylation by CKII is a critical event required for heterochromatin formation.
    DOI:  https://doi.org/10.1093/nar/gkae664
  26. Sci Adv. 2024 Aug 09. 10(32): eado2849
    Epigenetic reprogramming driving successful and failed repair in acute kidney injury.
    Yoshiharu Muto, Eryn E Dixon, Yasuhiro Yoshimura, Nicolas Ledru, Yuhei Kirita, Haojia Wu, Benjamin D Humphreys.
      Acute kidney injury (AKI) causes epithelial damage followed by subsequent repair. While successful repair restores kidney function, this process is often incomplete and can lead to chronic kidney disease (CKD) in a process called failed repair. To better understand the epigenetic reprogramming driving this AKI-to-CKD transition, we generated a single-nucleus multiomic atlas for the full mouse AKI time course, consisting of ~280,000 single-nucleus transcriptomes and epigenomes. We reveal cell-specific dynamic alterations in gene regulatory landscapes reflecting, especially, activation of proinflammatory pathways. We further generated single-nucleus multiomic data from four human AKI samples including validation by genome-wide identification of nuclear factor κB binding sites. A regularized regression analysis identifies key regulators involved in both successful and failed repair cell fate, identifying the transcription factor CREB5 as a regulator of both successful and failed tubular repair that also drives proximal tubular cell proliferation after injury. Our interspecies multiomic approach provides a foundation to comprehensively understand cell states in AKI.
    DOI:  https://doi.org/10.1126/sciadv.ado2849
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