bims-crepig 2024-12-15 papers

bims-crepig

Biomed News

on Chromatin regulation and epigenetics in cell fate and cancer

Issue of 2024–12–15
twenty-six papers selected by
Connor Rogerson, University of Cambridge

EOMES establishes mesoderm and endoderm differentiation potential through SWI/SNF-mediated global enhancer remodeling.
Interchromosomal contacts between regulatory regions trigger stable transgenerational epigenetic inheritance in Drosophila.
ALFIN-like proteins link histone H3K4me3 to H2A ubiquitination and coordinate diverse chromatin modifications in Arabidopsis.
Transcription processes compete with loop extrusion to homogenize promoter and enhancer dynamics.
Structural basis of H3K36 trimethylation by SETD2 during chromatin transcription.
A function of TPL/TBL1-type corepressors is to nucleate the assembly of the preinitiation complex.
The cells are all-right: Regulation of the Lefty genes by separate enhancers in mouse embryonic stem cells.
Optimized reporters for multiplexed detection of transcription factor activity.
Zelda is dispensable for Drosophila melanogaster histone gene regulation.
TBP bookmarks and preserves neural stem cell fate memory by orchestrating local chromatin architecture.
Canonical androgen response element motifs are tumor suppressive regulatory elements in the prostate.
The farnesoid X receptor activates transcription independently of RXR at non-canonical response elements.
Unified bursting strategies in ectopic and endogenous even-skipped expression patterns.
Enforced activation of the CREB/KDM2B axis prevents alcohol-induced embryonic developmental delay.
CRL3ARMC5 ubiquitin ligase and Integrator phosphatase form parallel mechanisms to control early stages of RNA Pol II transcription.
Piwi regulates the usage of alternative transcription start sites in the Drosophila ovary.
RNA binding by Periphilin plays an essential role in initiating silencing by the HUSH complex.
Central control of dynamic gene circuits governs T cell rest and activation.
LBFextract: Unveiling transcription factor dynamics from liquid biopsy data.
All eukaryotic SMC proteins induce a twist of -0.6 at each DNA loop extrusion step.
Structural and kinetic insights into tRNA promoter engagement by yeast general transcription factor TFIIIC.
Activation-derepression synergy enables a bHLH network to coordinate a signal-specific fate response.
Hypoxia increases methylated histones to prevent histone clipping and heterochromatin redistribution during Raf-induced senescence.
Predicting gene expression from histone marks using chromatin deep learning models depends on histone mark function, regulatory distance and cellular states.
Resolving the chromatin impact of mosaic variants with targeted Fiber-seq.
Nucleo-cytoplasmic environment modulates spatiotemporal p53 phase separation.

Dev Cell. 2024 Dec 05. pii: S1534-5807(24)00696-8. [Epub ahead of print]

EOMES establishes mesoderm and endoderm differentiation potential through SWI/SNF-mediated global enhancer remodeling.

Chiara M Schröder, Lea Zissel, Sophie-Luise Mersiowsky, Mehmet Tekman, Simone Probst, Katrin M Schüle, Sebastian Preissl, Oliver Schilling, H Th Marc Timmers, Sebastian J Arnold.

  Mammalian pluripotent cells first segregate into neuroectoderm (NE), or mesoderm and endoderm (ME), characterized by lineage-specific transcriptional programs and chromatin states. To date, the relationship between transcription factor activities and dynamic chromatin changes that guide cell specification remains ill-defined. In this study, we employ mouse embryonic stem cell differentiation toward ME lineages to reveal crucial roles of the Tbx factor Eomes to globally establish ME enhancer accessibility as the prerequisite for ME lineage competence and ME-specific gene expression. EOMES cooperates with the SWItch/sucrose non-fermentable (SWI/SNF) complex to drive chromatin rewiring that is essential to overcome default NE differentiation, which is favored by asymmetries in chromatin accessibility at pluripotent state. Following global ME enhancer remodeling, ME-specific gene transcription is controlled by additional signals such as Wnt and transforming growth factor β (TGF-β)/NODAL, as a second layer of gene expression regulation, which can be mechanistically separated from initial chromatin remodeling activities.

Keywords:  Eomes; SWI/SNF complex; Tbx factors; cell fate decision; chromatin accessibility; enhancer regulation; epigenetic remodeling; gastrulation

DOI:  https://doi.org/10.1016/j.devcel.2024.11.014
Mol Cell. 2024 Dec 03. pii: S1097-2765(24)00946-8. [Epub ahead of print]

Interchromosomal contacts between regulatory regions trigger stable transgenerational epigenetic inheritance in Drosophila.

Maximilian H Fitz-James, Gonzalo Sabarís, Peter Sarkies, Frédéric Bantignies, Giacomo Cavalli.

  Non-genetic information can be inherited across generations in a process known as transgenerational epigenetic inheritance (TEI). In Drosophila, hemizygosity of the Fab-7 regulatory element triggers inheritance of the histone mark H3K27me3 at a homologous locus on another chromosome, resulting in heritable epigenetic differences in eye color. Here, by mutating transcription factor binding sites within the Fab-7 element, we demonstrate the importance of the proteins pleiohomeotic and GAGA factor in the establishment and maintenance of TEI. We show that these proteins function by recruiting the polycomb repressive complex 2 and by mediating interchromosomal chromatin contacts between Fab-7 and its homologous locus, respectively. Using an in vivo synthetic biology system to induce them, we then show that chromatin contacts alone can establish TEI, providing a mechanism by which hemizygosity of one locus can establish epigenetic memory at another distant locus in trans through chromatin contacts.

Keywords:  3D genome organization; Fab-7; GAGA factor; chromatin contacts; epimutation; polycomb; transgenerational epigenetic inheritance

DOI:  https://doi.org/10.1016/j.molcel.2024.11.021
Mol Plant. 2024 Dec 11. pii: S1674-2052(24)00391-5. [Epub ahead of print]

ALFIN-like proteins link histone H3K4me3 to H2A ubiquitination and coordinate diverse chromatin modifications in Arabidopsis.

Xiao-Min Su, Dan-Yang Yuan, Na Liu, Zhao-Chen Zhang, Minqi Yang, Lin Li, She Chen, Yue Zhou, Xin-Jian He.

  Trimethylation of histone H3K4 (H3K4me3) is widely distributed at numerous actively transcribed protein-coding genes throughout the genome. However, the interplay between H3K4me3 and other chromatin modifications in plants remains poorly understood. In this study, we find that the Arabidopsis thaliana ALFIN-LIKE (AL) proteins contain a C-terminal PHD finger capable of binding to H3K4me3, along with a PHD-associated AL (PAL) domain that interacts with components of the Polycomb repressive complex 1 (PRC1), thereby facilitating H2A ubiquitination (H2Aub) at H3K4me3-enriched genes throughout the genome. Furthermore, we demonstrate that the loss of SDG2, a key histone H3K4 methyltransferase, leads to a reduction in H3K4me3 level, which subsequently causes a decrease in H2Aub on a genome-wide scale, revealing a strong association between H3K4me3 and H2Aub. Additionally, we show that the PAL domain interacts with various other chromatin-related proteins or complexes, including those involved in regulating H2A.Z deposition, H3K27me3 demethylation, histone deacetylation, and chromatin accessibility. Our genome-wide analysis suggests that the AL proteins play a crucial role in coordinating H3K4me3 with multiple other chromatin modifications across the genome.

Keywords:  ALFIN-like proteins; H2A.Z; H2Aub; H3K4me3; PRC1; histone

DOI:  https://doi.org/10.1016/j.molp.2024.12.007
Sci Adv. 2024 Dec 13. 10(50): eadq0987

Transcription processes compete with loop extrusion to homogenize promoter and enhancer dynamics.

Angeliki Platania, Cathie Erb, Mariano Barbieri, Bastien Molcrette, Erwan Grandgirard, Marit A C de Kort, Wim Pomp, Karen Meaburn, Tiegh Taylor, Virlana M Shchuka, Silvia Kocanova, Mariia Nazarova, Guilherme Monteiro Oliveira, Jennifer A Mitchell, Evi Soutoglou, Tineke L Lenstra, Nacho Molina, Argyris Papantonis, Kerstin Bystricky, Tom Sexton.

The spatiotemporal configuration of genes with distal regulatory elements is believed to be crucial for transcriptional control, but full mechanistic understanding is lacking. We combine simultaneous live tracking of pairs of genomic loci and nascent transcripts with molecular dynamics simulations to assess the Sox2 gene and its enhancer. We find that both loci exhibit more constrained mobility than control sequences due to stalled cohesin at CCCTC-binding factor sites. Strikingly, enhancer mobility becomes constrained on transcriptional firing, homogenizing its dynamics with the gene promoter, suggestive of their cotranscriptional sharing of a nuclear microenvironment. Furthermore, we find transcription and loop extrusion to be antagonistic processes constraining regulatory loci. These findings indicate that modulating chromatin mobility can be an additional, underestimated means for effective gene regulation.

DOI: https://doi.org/10.1126/sciadv.adq0987
Science. 2024 Dec 12. eadn6319

Structural basis of H3K36 trimethylation by SETD2 during chromatin transcription.

Jonathan W Markert, Jelly H Soffers, Lucas Farnung.

During transcription, RNA polymerase II traverses through chromatin, and post-translational modifications including histone methylations mark regions of active transcription. Histone protein H3 lysine 36 trimethylation (H3K36me3), which is established by the histone methyltransferase SETD2, suppresses cryptic transcription, regulates splicing, and serves as a binding site for transcription elongation factors. The mechanism by which the transcription machinery coordinates the deposition of H3K36me3 is not well understood. Here we provide cryo-electron microscopy structures of mammalian RNA polymerase II-DSIF-SPT6-PAF1c-TFIIS-IWS1-SETD2-nucleosome elongation complexes, revealing that the transcription machinery regulates H3K36me3 deposition by SETD2 on downstream and upstream nucleosomes. SPT6 binds the exposed H2A-H2B dimer during transcription and the SPT6 death-like domain mediates an interaction with SETD2 bound to a nucleosome upstream of RNA polymerase II.

DOI: https://doi.org/10.1126/science.adn6319
J Cell Biol. 2025 Feb 03. pii: e202404103. [Epub ahead of print]224(2):

A function of TPL/TBL1-type corepressors is to nucleate the assembly of the preinitiation complex.

Alexander R Leydon, Benjamin Downing, Janet Solano Sanchez, Raphael Loll-Krippleber, Nathan M Belliveau, Ricard A Rodriguez-Mias, Andrew J Bauer, Isabella J Watson, Lena Bae, Judit Villén, Grant W Brown, Jennifer L Nemhauser.

The plant corepressor TPL is recruited to diverse chromatin contexts, yet its mechanism of repression remains unclear. Previously, we leveraged the fact that TPL retains its function in a synthetic transcriptional circuit in the yeast model Saccharomyces cerevisiae to localize repressive function to two distinct domains. Here, we employed two unbiased whole-genome approaches to map the physical and genetic interactions of TPL at a repressed locus. We identified SPT4, SPT5, and SPT6 as necessary for repression with SPT4 acting as a bridge connecting TPL to SPT5 and SPT6. We discovered the association of multiple additional constituents of the transcriptional preinitiation complex at TPL-repressed promoters, specifically those involved early in transcription initiation. These findings were validated in yeast and plants, including a novel method to analyze the conditional loss of function of essential genes in plants. Our findings support a model where TPL nucleates preassembly of the transcription activation machinery to facilitate the rapid onset of transcription once repression is relieved.

DOI: https://doi.org/10.1083/jcb.202404103
PLoS Genet. 2024 Dec 13. 20(12): e1011513

The cells are all-right: Regulation of the Lefty genes by separate enhancers in mouse embryonic stem cells.

Tiegh Taylor, Hongyu Vicky Zhu, Sakthi D Moorthy, Nawrah Khader, Jennifer A Mitchell.

Enhancers play a critical role in regulating precise gene expression patterns essential for development and cellular identity; however, how gene-enhancer specificity is encoded within the genome is not clearly defined. To investigate how this specificity arises within topologically associated domains (TAD), we performed allele-specific genome editing of sequences surrounding the Lefty1 and Lefty2 paralogs in mouse embryonic stem cells. The Lefty genes arose from a tandem duplication event and these genes interact with each other in chromosome conformation capture assays which place these genes within the same TAD. Despite their physical proximity, we demonstrate that these genes are primarily regulated by separate enhancer elements. Through CRISPR-Cas9 mediated deletions to remove the intervening chromatin between the Lefty genes, we reveal a distance-dependent dosage effect of the Lefty2 enhancer on Lefty1 expression. These findings indicate a role for chromatin distance in insulating gene expression domains in the Lefty locus in the absence of architectural insulation.

DOI: https://doi.org/10.1371/journal.pgen.1011513
Cell Syst. 2024 Dec 03. pii: S2405-4712(24)00312-0. [Epub ahead of print]

Optimized reporters for multiplexed detection of transcription factor activity.

Max Trauernicht, Teodora Filipovska, Chaitanya Rastogi, Bas van Steensel.

  In any given cell type, dozens of transcription factors (TFs) act in concert to control the activity of the genome by binding to specific DNA sequences in regulatory elements. Despite their considerable importance, we currently lack simple tools to directly measure the activity of many TFs in parallel. Massively parallel reporter assays (MPRAs) allow the detection of TF activities in a multiplexed fashion; however, we lack basic understanding to rationally design sensitive reporters for many TFs. Here, we use an MPRA to systematically optimize transcriptional reporters for 86 TFs and evaluate the specificity of all reporters across a wide array of TF perturbation conditions. We thus identified critical TF reporter design features and obtained highly sensitive and specific reporters for 62 TFs, many of which outperform available reporters. The resulting collection of "prime" TF reporters can be used to uncover TF regulatory networks and to illuminate signaling pathways. A record of this paper's transparent peer review process is included in the supplemental information.

Keywords:  MPRA; TF; TF reporter assay; TF reporter design; massively parallel reporter assay; multiplexed TF reporter assay; reporter; signaling pathways; specificity; transcription factor

DOI:  https://doi.org/10.1016/j.cels.2024.11.003
Mol Biol Cell. 2024 Dec 11. mbcE24010028

Zelda is dispensable for Drosophila melanogaster histone gene regulation.

Tommy O'Haren, Tsutomu Aoki, Leila E Rieder.

To ensure that the embryo can package exponentially increasing amounts of DNA, replication-dependent histones are some of the earliest transcribed genes from the zygotic genome. However, how the histone genes are identified is not known. The Drosophila melanogaster pioneer factor CLAMP regulates the embryonic histone genes and helps establish the histone locus body, a suite of factors that controls histone mRNA biosynthesis, but CLAMP is not unique to the histone genes. Zelda collaborates with CLAMP across the genome to regulate zygotic genome activation and target early activated genes. We hypothesized that Zelda helps identify histone genes for early embryonic expression. We found that Zelda targets the histone gene locus early during embryogenesis, prior to histone gene expression. However, depletion of zelda in the early embryo does not affect histone mRNA levels or prevent the recruitment of other factors. These results suggest the earliest events responsible for specifying the zygotic histone genes remain undiscovered.

DOI: https://doi.org/10.1091/mbc.E24-01-0028
Mol Cell. 2024 Dec 05. pii: S1097-2765(24)00944-4. [Epub ahead of print]

TBP bookmarks and preserves neural stem cell fate memory by orchestrating local chromatin architecture.

Yuying Shen, Kun Liu, Jie Liu, Jingwen Shen, Tongtong Ye, Runxiang Zhao, Rulan Zhang, Yan Song.

  Mitotic bookmarking has been posited as an important strategy for cells to faithfully propagate their fate memory through cell generations. However, the physiological significance and regulatory mechanisms of mitotic bookmarking in neural development remain unexplored. Here, we identified TATA-binding protein (TBP) as a crucial mitotic bookmarker for preserving the fate memory of Drosophila neural stem cells (NSCs). Phosphorylation by the super elongation complex (SEC) is important for TBP to retain as discrete foci at mitotic chromosomes of NSCs to effectively transmit their fate memory. TBP depletion leads to drastic NSC loss, whereas TBP overexpression enhances the ability of SEC to induce neural progenitor dedifferentiation and tumorigenesis. Importantly, TBP achieves its mitotic retention through recruiting the chromatin remodeler EP400, which in turn increases local chromatin accessibility via depositing H2A.Z. Thus, local chromatin remodeling ensures mitotic bookmarking, which may represent a general principle underlying the preservation of cell fate memory.

Keywords:  Drosophila melanogaster; EP400; H2A.Z; TBP; cell fate memory; chromatin remodeler; mitotic bookmarking; mitotic retention; neural development; neural stem cell

DOI:  https://doi.org/10.1016/j.molcel.2024.11.019
Nat Commun. 2024 Dec 13. 15(1): 10675

Canonical androgen response element motifs are tumor suppressive regulatory elements in the prostate.

Xuanrong Chen, Michael A Augello, Deli Liu, Kevin Lin, Alex Hakansson, Martin Sjöström, Francesca Khani, Lesa D Deonarine, Yang Liu, Jaida Travascio-Green, Jiansheng Wu, Un In Chan, Jude Owiredu, Massimo Loda, Felix Y Feng, Brian D Robinson, Elai Davicioni, Andrea Sboner, Christopher E Barbieri.

The androgen receptor (AR) is central in prostate tissue identity and differentiation, and controls normal growth-suppressive, prostate-specific gene expression. It also drives prostate tumorigenesis when hijacked for oncogenic transcription. The execution of growth-suppressive AR transcriptional programs in prostate cancer (PCa) and the potential for reactivation remain unclear. Here, we use a genome-wide approach to modulate canonical androgen response element (ARE) motifs-the classic DNA binding elements for AR-to delineate distinct AR transcriptional programs. We find that activating these AREs promotes differentiation and growth-suppressive transcription, potentially leading to AR+ PCa cell death, while ARE repression is tolerated by PCa cells but deleterious to normal prostate cells. Gene signatures driven by ARE activity correlate with improved prognosis and luminal phenotypes in PCa patients. Canonical AREs maintain a normal, lineage-specific transcriptional program that can be reengaged in PCa cells, offering therapeutic potential and clinical relevance.

DOI: https://doi.org/10.1038/s41467-024-53734-z
Nucleic Acids Res. 2024 Dec 09. pii: gkae1214. [Epub ahead of print]

The farnesoid X receptor activates transcription independently of RXR at non-canonical response elements.

Suzanne W van der Veen, Jelmer J Dijkstra, Ellen C L Willemsen, René Houtman, Alexandra Milona, Nikolas Marchet, Maureen Spit, Danielle Hollman, Fried J T Zwartkruis, Michiel Vermeulen, Jose M Ramos Pittol, Saskia W C van Mil.

The farnesoid X receptor (FXR) is a nuclear receptor (NR) known to obligately heterodimerize with the retinoid X receptor (RXR). FXR is expressed as four isoforms (α1-α4) that drive transcription from IR-1 (inverted repeat-1) response elements (REs). Recently, we found that FXR isoforms α2/α4 also activate transcription from non-canonical ER-2 (everted repeat-2) REs, mediating most metabolic effects of general FXR activation. Here, we explored molecular determinants of regulation by FXRα2 from ER-2 REs through quantitative interaction proteomics, site-directed mutagenesis and transcriptomics. We discovered FXRα2 binds to and activates ER-2 elements in vitro and in reporter assays independently of RXR. Genome-wide binding analysis in mouse liver revealed higher ER-2 motif enrichment in FXR sites lacking RXR. Abrogation of FXRα2:RXR heterodimerization abolished IR-1, but preserved ER-2 transactivation. Transcriptome-wide, RXR overexpression inhibited 25% of FXRα2 targets in HepG2. These genes were specifically activated by the heterodimerization-deficient mutant FXRα2L434R, enriched for ER-2 motifs at their promoters, and involved in lipid metabolism and ammonia detoxification. In conclusion, RXR acts as a molecular switch, inhibiting FXRα2 activation from ER-2 while enhancing it from canonical IR-1 REs. Our results showcase FXR as the first NR with isoform-specific RXR-independent REs, highlighting a new layer of regulation and complexity for RXR-heterodimerizing NRs.

DOI: https://doi.org/10.1093/nar/gkae1214
Elife. 2024 Dec 09. pii: RP88671. [Epub ahead of print]12

Unified bursting strategies in ectopic and endogenous even-skipped expression patterns.

Augusto Berrocal, Nicholas C Lammers, Hernan G Garcia, Michael B Eisen.

  Transcription often occurs in bursts as gene promoters switch stochastically between active and inactive states. Enhancers can dictate transcriptional activity in animal development through the modulation of burst frequency, duration, or amplitude. Previous studies observed that different enhancers can achieve a wide range of transcriptional outputs through the same strategies of bursting control. For example, in Berrocal et al., 2020, we showed that despite responding to different transcription factors, all even-skipped enhancers increase transcription by upregulating burst frequency and amplitude while burst duration remains largely constant. These shared bursting strategies suggest that a unified molecular mechanism constraints how enhancers modulate transcriptional output. Alternatively, different enhancers could have converged on the same bursting control strategy because of natural selection favoring one of these particular strategies. To distinguish between these two scenarios, we compared transcriptional bursting between endogenous and ectopic gene expression patterns. Because enhancers act under different regulatory inputs in ectopic patterns, dissimilar bursting control strategies between endogenous and ectopic patterns would suggest that enhancers adapted their bursting strategies to their trans-regulatory environment. Here, we generated ectopic even-skipped transcription patterns in fruit fly embryos and discovered that bursting strategies remain consistent in endogenous and ectopic even-skipped expression. These results provide evidence for a unified molecular mechanism shaping even-skipped bursting strategies and serve as a starting point to uncover the realm of strategies employed by other enhancers.

Keywords:  D. melanogaster; chromosomes; development; developmental biology; enhancers; gene expression; imaging; patterning; transcription

DOI:  https://doi.org/10.7554/eLife.88671
Cell Rep. 2024 Dec 10. pii: S2211-1247(24)01426-8. [Epub ahead of print]43(12): 115075

Enforced activation of the CREB/KDM2B axis prevents alcohol-induced embryonic developmental delay.

Hang Liu, Qiyu Ren, Meihan Gong, Feifei Zuo, Qian Li, Dawei Huo, Ye Yuan, Yutong Zhang, Yu Kong, Xiaozhi Liu, Cailing Lu, Xudong Wu.

  Unintentional, early pregnancy alcohol consumption affects embryonic development. During the peri-implantation stage, coinciding with the transition from naive to primed pluripotency, the long isoform of KDM2B (KDM2BLF) underlies the de novo establishment of polycomb repressive complex (PRC) functions at promoters after fertilization. However, it remains unclear whether and how ethanol exposure affects this spatiotemporal chromatin setting. Here, we show that exposing peri-implantation mouse embryos to ethanol leads to impaired post-implantation development, mirrored by the delayed exit of naive pluripotency in acetaldehyde-treated embryonic stem cells. Remarkably, these abnormalities are linked to inadequate KDM2BLF expression and compromised deposition of PRC marks, which arise from cAMP response element-binding protein (CREB) inactivation. Accordingly, pharmacological activation of CREB effectively restores pluripotency transition partly dependent on KDM2BLF in vitro and ameliorates post-implantation embryonic defects in vivo. Therefore, our study highlights the pivotal role of the CREB/KDM2B axis in chromatin configuration and developmental programming, proposing potential preventive strategies against ethanol exposure-induced detrimental effects.

Keywords:  CP: Developmental biology; CP: Molecular biology; CREB; H3K27me3; KDM2B; PDE4 inhibitor; PRC2; acetaldehyde; ethanol exposure; pluripotency; polycomb; post-implantation

DOI:  https://doi.org/10.1016/j.celrep.2024.115075
Mol Cell. 2024 Dec 05. pii: S1097-2765(24)00949-3. [Epub ahead of print]

CRL3ARMC5 ubiquitin ligase and Integrator phosphatase form parallel mechanisms to control early stages of RNA Pol II transcription.

Roberta Cacioppo, Alexander Gillis, Iván Shlamovitz, Andrew Zeller, Daniela Castiblanco, Alastair Crisp, Benjamin Haworth, Angela Arabiotorre, Pegah Abyaneh, Yu Bao, Julian E Sale, Scott Berry, Ana Tufegdžić Vidaković.

  Control of RNA polymerase II (RNA Pol II) through ubiquitylation is essential for the DNA-damage response. Here, we reveal a distinct ubiquitylation pathway in human cells, mediated by CRL3ARMC5, that targets excessive and defective RNA Pol II molecules at the initial stages of the transcription cycle. Upon ARMC5 loss, RNA Pol II accumulates in the free pool and in the promoter-proximal zone but is not permitted into elongation. We identify Integrator subunit 8 (INTS8) as a gatekeeper preventing the release of excess RNA Pol II molecules into gene bodies. Combined loss of ARMC5 and INTS8 has detrimental effects on cell growth and results in the uncontrolled release of excessive RNA Pol II complexes into early elongation, many of which are transcriptionally incompetent and fail to reach the ends of genes. These findings uncover CRL3ARMC5 and Integrator as two distinct pathways acting in parallel to monitor the quantity and quality of transcription complexes before they are licensed into elongation.

Keywords:  ARMC5; CUL3; INTS8; Integrator; RNA polymerase II; elongation; pausing; promoter-proximal; transcription; ubiquitin

DOI:  https://doi.org/10.1016/j.molcel.2024.11.024
Nucleic Acids Res. 2024 Dec 09. pii: gkae1160. [Epub ahead of print]

Piwi regulates the usage of alternative transcription start sites in the Drosophila ovary.

Jiaying Chen, Na Liu, Hongying Qi, Nils Neuenkirchen, Yuedong Huang, Haifan Lin.

Alternative transcription initiation, which refers to the transcription of a gene from different transcription start sites (TSSs), is prevalent across metazoans and has important biological functions. Although transcriptional regulation has been extensively studied, the mechanism that selects one TSS over others within a gene remains elusive. Using the Cap Analysis of Gene Expression sequencing (CAGE-seq) method, we discovered that Piwi, an RNA-binding protein, regulates TSS usage in at least 87 genes. In piwi-deficient Drosophila ovaries, these genes displayed significantly altered TSS usage (ATU). The regulation of TSS usage occurred in both germline and somatic cells in ovaries, as well as in cultured ovarian somatic cells (OSCs). Correspondingly, RNA Polymerase II (Pol II) initiation and elongation at the TSSs of ATU genes were affected in germline-piwi-knockdown ovaries and piwi-knockdown OSCs. Furthermore, we identified a Facilitates Chromatin Transcription (FACT) complex component, Ssrp, that is essential for mRNA elongation, as a novel interactor of Piwi in the nucleus. Temporally controlled knockdown of ssrp affected TSS usage in ATU genes, whereas overexpression of ssrp partially rescued the TSS usage of ATU genes in piwi mutant ovaries. Thus, Piwi may interact with Ssrp to regulate TSS usage in Drosophila ovaries by affecting Pol II initiation and elongation.

DOI: https://doi.org/10.1093/nar/gkae1160
Nucleic Acids Res. 2024 Dec 11. pii: gkae1165. [Epub ahead of print]

RNA binding by Periphilin plays an essential role in initiating silencing by the HUSH complex.

Stuart Bloor, Niek Wit, Paul J Lehner.

The human silencing hub (HUSH) complex is a transcription-dependent, epigenetic repressor complex that provides a genome-wide immunosurveillance system for the recognition and silencing of newly-integrated retroelements. The core HUSH complex of TASOR, MPP8 and Periphilin, represses these retroelements through SETDB1-mediated H3K9me3 deposition and MORC2-dependent chromatin compaction. HUSH-dependent silencing is RNA-mediated, yet no HUSH component contains a recognised RNA-binding domain. Here we used an unbiased approach to identify which HUSH component was able to bind RNA and determine whether RNA-binding was essential for HUSH function. We identify Periphilin as the major RNA-binding component of the HUSH complex and show that Periphilin's N-terminal domain is essential for both RNA binding and HUSH function. Periphilin binding to RNA was independent of its interaction with TASOR or MPP8, as its N-terminal domain was sufficient for RNA targeting. The artificial tethering of Periphilin to a HUSH-insensitive, nascent transcript, enabled the HUSH-dependent silencing of the transcript. This tethering of Periphilin allowed the RNA-binding region of Periphilin to be removed such that only its C-terminal domain was required for oligomerisation and interaction with TASOR. We therefore show that Periphilin is the predominant RNA-binding protein of the HUSH complex and this RNA-binding is essential for HUSH activity.

DOI: https://doi.org/10.1093/nar/gkae1165
Nature. 2024 Dec 11.

Central control of dynamic gene circuits governs T cell rest and activation.

Maya M Arce, Jennifer M Umhoefer, Nadia Arang, Sivakanthan Kasinathan, Jacob W Freimer, Zachary Steinhart, Haolin Shen, Minh T N Pham, Mineto Ota, Anika Wadhera, Rama Dajani, Dmytro Dorovskyi, Yan Yi Chen, Qi Liu, Yuan Zhou, Danielle L Swaney, Kirsten Obernier, Brian R Shy, Julia Carnevale, Ansuman T Satpathy, Nevan J Krogan, Jonathan K Pritchard, Alexander Marson.

The ability of cells to maintain distinct identities and respond to transient environmental signals requires tightly controlled regulation of gene networks1-3. These dynamic regulatory circuits that respond to extracellular cues in primary human cells remain poorly defined. The need for context-dependent regulation is prominent in T cells, where distinct lineages must respond to diverse signals to mount effective immune responses and maintain homeostasis4-8. Here we performed CRISPR screens in multiple primary human CD4+ T cell contexts to identify regulators that control expression of IL-2Rα, a canonical marker of T cell activation transiently expressed by pro-inflammatory effector T cells and constitutively expressed by anti-inflammatory regulatory T cells where it is required for fitness9-11. Approximately 90% of identified regulators of IL-2Rα had effects that varied across cell types and/or stimulation states, including a subset that even had opposite effects across conditions. Using single-cell transcriptomics after pooled perturbation of context-specific screen hits, we characterized specific factors as regulators of overall rest or activation and constructed state-specific regulatory networks. MED12 - a component of the Mediator complex - serves as a dynamic orchestrator of key regulators, controlling expression of distinct sets of regulators in different T cell contexts. Immunoprecipitation-mass spectrometry revealed that MED12 interacts with the histone methylating COMPASS complex. MED12 was required for histone methylation and expression of genes encoding key context-specific regulators, including the rest maintenance factor KLF2 and the versatile regulator MYC. CRISPR ablation of MED12 blunted the cell-state transitions between rest and activation and protected from activation-induced cell death. Overall, this work leverages CRISPR screens performed across conditions to define dynamic gene circuits required to establish resting and activated T cell states.

DOI: https://doi.org/10.1038/s41586-024-08314-y
Comput Struct Biotechnol J. 2024 Dec;23 3163-3174

LBFextract: Unveiling transcription factor dynamics from liquid biopsy data.

Isaac Lazzeri, Benjamin Gernot Spiegl, Samantha O Hasenleithner, Michael R Speicher, Martin Kircher.

   Motivation: The analysis of circulating cell-free DNA (cfDNA) holds immense promise as a non-invasive diagnostic tool across various human conditions. However, extracting biological insights from cfDNA fragments entails navigating complex and diverse bioinformatics methods, encompassing not only DNA sequence variation, but also epigenetic characteristics like nucleosome footprints, fragment length, and methylation patterns.
Results: We introduce Liquid Biopsy Feature extract (LBFextract), a comprehensive package designed to streamline feature extraction from cfDNA sequencing data, with the aim of enhancing the reproducibility and comparability of liquid biopsy studies. LBFextract facilitates the integration of preprocessing and postprocessing steps through alignment fragment tags and a hook mechanism. It incorporates various methods, including coverage-based and fragment length-based approaches, alongside two novel feature extraction methods: an entropy-based method to infer TF activity from fragmentomics data and a technique to amplify signals from nucleosome dyads. Additionally, it implements a method to extract condition-specific differentially active TFs based on these features for biomarker discovery. We demonstrate the use of LBFextract for the subtype classification of advanced prostate cancer patients using coverage signals at transcription factor binding sites from cfDNA. We show that LBFextract can generate robust and interpretable features that can discriminate between different clinical groups. LBFextract is a versatile and user-friendly package that can facilitate the analysis and interpretation of liquid biopsy data.
Data and Code Availability and Implementation: LBFextract is freely accessible at https://github.com/Isy89/LBF. It is implemented in Python and compatible with Linux and Mac operating systems. Code and data to reproduce these analyses have been uploaded to 10.5281/zenodo.10964406.

Keywords:  Bioinformatics; Cell-free DNA; Fragmentomics; Transcription factors; Whole-genome sequencing

DOI:  https://doi.org/10.1016/j.csbj.2024.08.007
Sci Adv. 2024 Dec 13. 10(50): eadt1832

All eukaryotic SMC proteins induce a twist of -0.6 at each DNA loop extrusion step.

Richard Janissen, Roman Barth, Iain F Davidson, Jan-Michael Peters, Cees Dekker.

Eukaryotes carry three types of structural maintenance of chromosome (SMC) protein complexes, condensin, cohesin, and SMC5/6, which are ATP-dependent motor proteins that remodel the genome via DNA loop extrusion (LE). SMCs modulate DNA supercoiling but remains incompletely understood how this is achieved. Using a single-molecule magnetic tweezers assay that directly measures how much twist is induced by individual SMCs in each LE step, we demonstrate that all three SMC complexes induce the same large negative twist (i.e., linking number change [Formula: see text] of ~-0.6 at each LE step) into the extruded loop, independent of step size and DNA tension. Using ATP hydrolysis mutants and nonhydrolyzable ATP analogs, we find that ATP binding is the twist-inducing event during the ATPase cycle, coinciding with the force-generating LE step. The fact that all three eukaryotic SMC proteins induce the same amount of twist indicates a common DNA-LE mechanism among these SMC complexes.

DOI: https://doi.org/10.1126/sciadv.adt1832
Nucleic Acids Res. 2024 Dec 09. pii: gkae1174. [Epub ahead of print]

Structural and kinetic insights into tRNA promoter engagement by yeast general transcription factor TFIIIC.

Wolfram Seifert-Dávila, Anastasiia Chaban, Florence Baudin, Mathias Girbig, Luis Hauptmann, Thomas Hoffmann, Olivier Duss, Sebastian Eustermann, Christoph W Müller.

Transcription of transfer RNA (tRNA) genes by RNA polymerase (Pol) III requires the general transcription factor IIIC (TFIIIC), which recognizes intragenic A-box and B-box DNA motifs of type II gene promoters. However, the underlying mechanism has remained elusive, in part due to missing structural information for A-box recognition. In this study, we use single-particle cryogenic electron microscopy (cryo-EM) and single-molecule fluorescence resonance energy transfer (smFRET) to reveal structural and real-time kinetic insights into how the 520-kDa yeast TFIIIC complex engages A-box and B-box DNA motifs in the context of a tRNA gene promoter. Cryo-EM structures of τA and τB subcomplexes bound to the A-box and B-box were obtained at 3.7 and 2.5 Å resolution, respectively, while cryo-EM single-particle mapping determined the specific distance and relative orientation of the τA and τB subcomplexes revealing a fully engaged state of TFIIIC. smFRET experiments show that overall recruitment and residence times of TFIIIC on a tRNA gene are primarily governed by B-box recognition, while footprinting experiments suggest a key role of τA and the A-box in TFIIIB and Pol III recruitment following TFIIIC recognition of type II promoters.

DOI: https://doi.org/10.1093/nar/gkae1174
Cell Rep. 2024 Dec 12. pii: S2211-1247(24)01428-1. [Epub ahead of print]43(12): 115077

Activation-derepression synergy enables a bHLH network to coordinate a signal-specific fate response.

Sandy Nandagopal, Alexsandra Terrio, Fernando Z Vicente, Sean G Megason, Ashwini Jambhekar, Galit Lahav.

  Stem cells integrate multiple environmental signals to activate appropriate fate programs. To ensure coherent responses, alternative fates must be concomitantly inactivated. However, mechanisms that coordinate fates in a signal-specific manner are not fully understood. Here, we investigate the role of a network of basic-helix-loop-helix (bHLH) transcription factors in neural stem cells, which integrate leukemia inhibitory factor (LIF) and bone morphogenetic protein (BMP) signaling to synergistically induce glial fibrillary acidic protein (GFAP), a key astrocyte-fate determinant. Using quantitative RNA-fluorescence in situ hybridization (FISH) and ectopic expression, we find that multiple bHLHs that promote alternative fates also repress GFAP but are all suppressed by BMP and, to a lesser extent, LIF. Mathematical modeling shows that synergy arises from this coordinated derepression of GFAP combined with its activation by LIF signaling. Finally, we determine how coordinated and tunable derepression results from extensive cross-regulation among bHLHs. Activation-derepression synergy could be broadly utilized to couple signaling and fate, particularly across the numerous developmental systems regulated by bHLH factors.

Keywords:  CP: Molecular biology; GFAP; astrocyte; bHLH; cell fate; gene regulatory network; neural stem cell; signal integration; stem cell differentiation; synergy; transcription factor network

DOI:  https://doi.org/10.1016/j.celrep.2024.115077
Nucleic Acids Res. 2024 Dec 11. pii: gkae1210. [Epub ahead of print]

Hypoxia increases methylated histones to prevent histone clipping and heterochromatin redistribution during Raf-induced senescence.

Soojeong Chang, Ramhee Moon, Dowoon Nam, Sang-Won Lee, Insoo Yoon, Dong-Sung Lee, Seunghyuk Choi, Eunok Paek, Daehee Hwang, Junho K Hur, Youhyun Nam, Rakwoo Chang, Hyunsung Park.

Hypoxia enhances histone methylation by inhibiting oxygen- and α-ketoglutarate-dependent demethylases, resulting in increased methylated histones. This study reveals how hypoxia-induced methylation affects histone clipping and the reorganization of heterochromatin into senescence-associated heterochromatin foci (SAHF) during oncogene-induced senescence (OIS) in IMR90 human fibroblasts. Notably, using top-down proteomics, we discovered specific cleavage sites targeted by Cathepsin L (CTSL) in H3, H2B and H4 during Raf activation, identifying novel sites in H2B and H4. Hypoxia counteracts CTSL-mediated histone clipping by promoting methylation without affecting CTSL's activity. This increase in methylation under hypoxia protects against clipping, reshaping the epigenetic landscape and influencing chromatin accessibility, as shown by ATAC-seq analysis. These insights underscore the pivotal role of hypoxia-induced histone methylation in protecting chromatin from significant epigenetic shifts during cellular aging.

DOI: https://doi.org/10.1093/nar/gkae1210
Nucleic Acids Res. 2024 Dec 11. pii: gkae1212. [Epub ahead of print]

Predicting gene expression from histone marks using chromatin deep learning models depends on histone mark function, regulatory distance and cellular states.

Alan E Murphy, Aydan Askarova, Boris Lenhard, Nathan G Skene, Sarah J Marzi.

To understand the complex relationship between histone mark activity and gene expression, recent advances have used in silico predictions based on large-scale machine learning models. However, these approaches have omitted key contributing factors like cell state, histone mark function or distal effects, which impact the relationship, limiting their findings. Moreover, downstream use of these models for new biological insight is lacking. Here, we present the most comprehensive study of this relationship to date - investigating seven histone marks in eleven cell types across a diverse range of cell states. We used convolutional and attention-based models to predict transcription from histone mark activity at promoters and distal regulatory elements. Our work shows that histone mark function, genomic distance and cellular states collectively influence a histone mark's relationship with transcription. We found that no individual histone mark is consistently the strongest predictor of gene expression across all genomic and cellular contexts. This highlights the need to consider all three factors when determining the effect of histone mark activity on transcriptional state. Furthermore, we conducted in silico histone mark perturbation assays, uncovering functional and disease related loci and highlighting frameworks for the use of chromatin deep learning models to uncover new biological insight.

DOI: https://doi.org/10.1093/nar/gkae1212
Genome Res. 2024 Dec 09.

Resolving the chromatin impact of mosaic variants with targeted Fiber-seq.

Stephanie C Bohaczuk, Zachary J Amador, Chang Li, Benjamin J Mallory, Elliott G Swanson, Jane Ranchalis, Mitchell R Vollger, Katherine M Munson, Tom Walsh, Morgan O Hamm, Yizi Mao, Andre Lieber, Andrew B Stergachis.

Accurately quantifying the functional consequences of noncoding mosaic variants requires the pairing of DNA sequences with both accessible and closed chromatin architectures along individual DNA molecules-a pairing that cannot be achieved using traditional fragmentation-based chromatin assays. We demonstrate that targeted single-molecule chromatin fiber sequencing (Fiber-seq) achieves this, permitting single-molecule, long-read genomic, and epigenomic profiling across targeted >100 kb loci with ∼10-fold enrichment over untargeted sequencing. Targeted Fiber-seq reveals that pathogenic expansions of the DMPK CTG repeat that underlie Myotonic Dystrophy 1 are characterized by somatic instability and disruption of multiple nearby regulatory elements, both of which are repeat length-dependent. Furthermore, we reveal that therapeutic adenine base editing of the segmentally duplicated γ-globin (HBG1/HBG2) promoters in primary human hematopoietic cells induced toward an erythroblast lineage increases the accessibility of the HBG1 promoter as well as neighboring regulatory elements. Overall, we find that these non-protein coding mosaic variants can have complex impacts on chromatin architectures, including extending beyond the regulatory element harboring the variant.

DOI: https://doi.org/10.1101/gr.279747.124
Sci Adv. 2024 Dec 13. 10(50): eads0427

Nucleo-cytoplasmic environment modulates spatiotemporal p53 phase separation.

Debalina Datta, Ambuja Navalkar, Arunima Sakunthala, Ajoy Paul, Komal Patel, Shalaka Masurkar, Laxmikant Gadhe, Shouvik Manna, Arpita Bhattacharyya, Shinjinee Sengupta, Manisha Poudyal, Jyoti Devi, Ajay Singh Sawner, Pradeep Kadu, Ranjit Shaw, Satyaprakash Pandey, Semanti Mukherjee, Nitisha Gahlot, Kundan Sengupta, Samir K Maji.

Liquid-liquid phase separation of various transcription factors into biomolecular condensates plays an essential role in gene regulation. Here, using cellular models and in vitro studies, we show the spatiotemporal formation and material properties of p53 condensates that might dictate its function. In particular, p53 forms liquid-like condensates in the nucleus of cells, which can bind to DNA and perform transcriptional activity. However, cancer-associated mutations promote misfolding and partially rigidify the p53 condensates with impaired DNA binding ability. Irrespective of wild-type and mutant forms, the partitioning of p53 into cytoplasm leads to the condensate formation, which subsequently undergoes rapid solidification. In vitro studies show that abundant nuclear components such as RNA and nonspecific DNA promote multicomponent phase separation of the p53 core domain and maintain their liquid-like property, whereas specific DNA promotes its dissolution into tetrameric functional p53. This work provides mechanistic insights into how the life cycle and DNA binding properties of p53 might be regulated by phase separation.

DOI: https://doi.org/10.1126/sciadv.ads0427