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Spt5 is a well-conserved factor that manipulates multiple stages of transcription from promoter-proximal pausing (PPP) to termination. Recent studies have revealed an unexpected increase of antisense transcripts near promoters in cells expressing mutant Spt5. Here, we identify Spt5p-restricted intragenic antisense transcripts and their close relationship with sense transcription in yeast. We confirm that Spt5 CTR phosphorylation is also important to retain Spt5's facility to regulate antisense transcription. The genes whose antisense transcription is strongly suppressed by Spt5p share strong endogenous sense transcription and weak antisense transcription, and this pattern is conserved in humans. Mechanistically, we found that Spt5p depletion increased histone acetylation to initiate intragenic antisense transcription by altering chromatin structure. We additionally identified termination factors that appear to be involved in the ability of Spt5p to restrict antisense transcription. By unveiling a new role of Spt5 in finely balancing the bidirectionality of transcription, we demonstrate that Spt5-mediated suppression of DSIF complex regulated-unstable transcripts (DUTs) is essential to sustain the accurate transcription by RNA polymerase II., (© 2024. The Author(s).)

Nitric oxide (·NO) is one of the toxic metabolites that bacteria can be exposed to within phagosomes. Gre factors, which are also known as transcript cleavage factors or transcription elongation factors, relieve back-tracked transcription elongation complexes by cleaving nascent RNAs, which allows transcription to resume after stalling. Here we discovered that loss of both Gre factors in Escherichia coli , GreA and GreB, significantly compromised ·NO detoxification due to ·NO-induced phenotypic heterogeneity in Δ greA Δ greB populations, which did not occur in wild-type cultures. Under normal culturing conditions, both wild-type and Δ greA Δ greB synthesized transcripts uniformly, whereas treatment with ·NO led to bimodal transcript levels in Δ greA Δ greB that were unimodal in wild-type. Interestingly, exposure to another toxic metabolite of phagosomes, hydrogen peroxide (H 2 O 2 ), produced analogous results. Furthermore, we showed that loss of Gre factors led to cheating under ·NO stress where transcriptionally deficient cells benefited from the detoxification activities of the transcriptionally proficient subpopulation. Collectively, these results show that loss of Gre factor activities produces phenotypic heterogeneity under ·NO and H 2 O 2 stress that can yield cheating between subpopulations.IMPORTANCEToxic metabolite stress occurs in a broad range of contexts that are important to human health, microbial ecology, and biotechnology, whereas Gre factors are highly conserved throughout the bacterial kingdom. Here we discovered that loss of Gre factors in E. coli leads to phenotypic heterogeneity under ·NO and H 2 O 2 stress, which we further show with ·NO results in cheating between subpopulations. Collectively, these data suggest that Gre factors play a role in coping with toxic metabolite stress, and that loss of Gre factors can produce cheating between neighbors., Competing Interests: The authors declare no conflict of interest.

Key Message: Barley reproductive fitness and efficient heat stress adaptation requires the activity of TFIIS, the elongation cofactor of RNAPII. Regulation of transcriptional machinery and its adaptive role under different stress conditions are studied extensively in the dicot model plant Arabidopsis, but our knowledge on monocot species remains elusive. TFIIS is an RNA polymerase II-associated transcription elongation cofactor. Previously, it was shown that TFIIS ensures efficient transcription elongation that is necessary for heat stress survival in A. thaliana. However, the function of TFIIS has not been analysed in monocots. In the present work, we have generated and studied independent tfIIs-crispr-mutant barley lines. We show that TFIIS is needed for reproductive development and heat stress survival in barley. The molecular basis of HS-sensitivity of tfIIs mutants is the retarded expression of heat stress protein transcripts, which leads to late accumulation of HSP chaperones, enhanced proteotoxicity and ultimately to lethality. We also show that TFIIS is transcriptionally regulated in response to heat, supporting a conserved adaptive function of these control elements for plant thermal adaptation. In sum, our results are a step forward for the better understanding of transcriptional machinery regulation in monocot crops., (© 2024. The Author(s).)

RNA polymerase (RNAP), the central enzyme of transcription, intermittently pauses during the elongation stage of RNA synthesis. Pausing provides an opportunity for regulatory events such as nascent RNA folding or the recruitment of transregulators. NusG (Spt5 in eukaryotes and archaea) regulates RNAP pausing and is the only transcription factor conserved across all cellular life. NusG is a multifunctional protein: its N-terminal domain (NGN) binds to RNAP, and its C-terminal KOW domain in bacteria interacts with transcription regulators such as ribosomes and termination factors. In Escherichia coli, NusG acts as an antipausing factor. However, recent studies have revealed that NusG has distinct transcriptional regulatory roles specific to bacterial clades with clinical implications. Here, we focus on NusG's dual roles in the regulation of pausing., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

In this issue of Molecular Cell, Engeholm et al. 1 present cryo-EM structures of the chromatin remodeler Chd1 bound to a hexasome-nucleosome complex, an intermediate state during transcription either with or without FACT to restore the missing H2A-H2B dimer. These two binding modes reveal how Chd1 and FACT cooperate in nucleosome re-establishment during transcription., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

To maintain the nucleosome organization of transcribed genes, ATP-dependent chromatin remodelers collaborate with histone chaperones. Here, we show that at the 5' ends of yeast genes, RNA polymerase II (RNAPII) generates hexasomes that occur directly adjacent to nucleosomes. The resulting hexasome-nucleosome complexes are then resolved by Chd1. We present two cryoelectron microscopy (cryo-EM) structures of Chd1 bound to a hexasome-nucleosome complex before and after restoration of the missing inner H2A/H2B dimer by FACT. Chd1 uniquely interacts with the complex, positioning its ATPase domain to shift the hexasome away from the nucleosome. In the absence of the inner H2A/H2B dimer, its DNA-binding domain (DBD) packs against the ATPase domain, suggesting an inhibited state. Restoration of the dimer by FACT triggers a rearrangement that displaces the DBD and stimulates Chd1 remodeling. Our results demonstrate how chromatin remodelers interact with a complex nucleosome assembly and suggest how Chd1 and FACT jointly support transcription by RNAPII., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Faithful transfer of parental histones to newly replicated daughter DNA strands is critical for inheritance of epigenetic states. Although replication proteins that facilitate parental histone transfer have been identified, how intact histone H3-H4 tetramers travel from the front to the back of the replication fork remains unknown. Here, we use AlphaFold-Multimer structural predictions combined with biochemical and genetic approaches to identify the Mrc1/CLASPIN subunit of the replisome as a histone chaperone. Mrc1 contains a conserved histone-binding domain that forms a brace around the H3-H4 tetramer mimicking nucleosomal DNA and H2A-H2B histones, is required for heterochromatin inheritance, and promotes parental histone recycling during replication. We further identify binding sites for the FACT histone chaperone in Swi1/TIMELESS and DNA polymerase α that are required for heterochromatin inheritance. We propose that Mrc1, in concert with FACT acting as a mobile co-chaperone, coordinates the distribution of parental histones to newly replicated DNA., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

LoaP is a member of the universal NusG protein family. Previously, we reported that unlike other characterized homologs, LoaP binds RNA sequence-specifically, recognizing a stem-loop in the 5'-untranslated region of operons it regulates. To elucidate how this NusG homolog acquired this ability, we now determined the co-crystal structure of Thermoanaerobacter pseudethanolicus LoaP bound to its cognate 26-nucleotide dfn RNA element. Our structure reveals that the LoaP C-terminal KOW domain recognizes the helical portion of the RNA by docking into a broadened major groove, while a protruding β-hairpin of the N-terminal NusG-like domain binds the UNCG tetraloop capping the stem-loop. Major-groove RNA recognition is unusual and is made possible by conserved features of the dfn hairpin. Superposition with structures of other NusG proteins implies that LoaP can bind concurrently to the dfn RNA and the transcription elongation complex, suggesting a new level of co-transcriptional regulation by proteins of this conserved family., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Histone acylation plays a pivotal role in modulating gene expression, ensuring proper neurogenesis and responsiveness to various signals. Recently, the evolutionary conserved YAF9, ENL, AF9, TAF41, SAS5 (YEATS) domain found in four human paralogs, has emerged as a new class of histone acylation reader with a preference for the bulkier crotonyl group lysine over acetylation. Despite advancements, the role of either histone crotonylation or its readers in neurons remains unclear. In this study, we employed Drosophila melanogaster to investigate the role of ENL/AF9 (dENL/AF9) in the nervous system. Pan-neuronal dENL/AF9 knockdown not only extended the lifespan of flies but also enhanced their overall fitness during aging, including improved sleep quality and locomotion. Moreover, a decreased activity of dENL/AF9 in neurons led to an up-regulation of catalase gene expression which combined with reduced levels of malondialdehyde (MDA) and an enhanced tolerance to oxidative stress in aging flies. This study unveiled a novel function of histone crotonylation readers in aging with potential implications for understanding age-related conditions in humans., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

After an RNA polymerase reaches a terminator, instead of dissociating from the template, it may diffuse along the DNA and recommence RNA synthesis from the previous or a different promoter. Magnetic tweezers were used to monitor such secondary transcription and determine the effects of low forces assisting or opposing translocation, protein roadblocks, and transcription factors. Remarkably, up to 50% of Escherichia coli (E. coli) RNA polymerases diffused along the DNA after termination. Force biased the direction of diffusion (sliding) and the velocity increased rapidly with force up to 0.7 pN and much more slowly thereafter. Sigma factor 70 (σ 70 ) likely remained associated with the DNA promoting sliding and enabling re-initiation from promoters in either orientation. However, deletions of the α-C-terminal domains severely limited the ability of RNAP to turn around between successive rounds of transcription. The addition of elongation factor NusG, which competes with σ 70 for binding to RNAP, limited additional rounds of transcription. Surprisingly, sliding RNA polymerases blocked by a DNA-bound lac repressor could slowly re-initiate transcription and were not affected by NusG, suggesting a σ-independent pathway. Low forces effectively biased promoter selection suggesting a prominent role for topological entanglements that affect RNA polymerase translocation., (© 2024. The Author(s).)

The histone acylation reader eleven-nineteen leukemia (ENL) plays a pivotal role in sustaining oncogenesis in acute leukemias, particularly in mixed-lineage leukemia -rearranged ( MLL -r) leukemia. ENL relies on its reader domain to recognize histone lysine acylation promoting oncogenic gene expression and leukemia progression. Here, we report the development of MS41, a highly potent and selective von Hippel-Lindau-recruiting ENL degrader that effectively inhibits the growth of ENL-dependent leukemia cells. MS41-induced ENL degradation reduces the chromatin occupancy of ENL-associated transcription elongation machinery, resulting in the suppression of key oncogenic gene expression programs and the activation of differentiation genes. MS41 is well-tolerated in vivo and substantially suppresses leukemia progression in a xenograft mouse model of MLL-r leukemia. Notably, MS41 also induces the degradation of mutant ENL proteins identified in Wilms' tumors. Our findings emphasize the therapeutic potential of pharmacological ENL degradation for treating ENL-dependent cancers, making MS41 not only a valuable chemical probe but also potential anticancer therapeutic for further development.

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

It is well known that modifiers play a role in ameliorating or exacerbating disease phenotypes in patients and carriers of recessively inherited disorders such as sickle cell disease and thalassemia. Here, we give an overview of the literature concerning a recently described association in carriers of SUPT5H Loss-of-Function variants with a beta-thalassemia-like phenotype including the characteristic elevated levels of HbA 2 . That SUPT5H acts as modifier in beta-thalassemia carriers became evident from three reported cases in whom combined heterozygosity of SUPT5H and HBB gene variants was observed to resemble a mild beta-thalassemia intermedia phenotype. The different SUPT5H variants and hematologic parameters reported are collected and reviewed to provide insight into the possible effects on hematologic expression, as well as potential disease mechanisms in carriers and patients.

RNA polymerase II (RNA Pol II)-mediated transcription is a critical, highly regulated process aided by protein complexes at distinct steps. Here, to investigate RNA Pol II and transcription-factor-binding and dissociation dynamics, we generated endogenous photoactivatable-GFP (PA-GFP) and HaloTag knockins using CRISPR-Cas9, allowing us to track a population of molecules at the induced Hsp70 loci in Drosophila melanogaster polytene chromosomes. We found that early in the heat-shock response, little RNA Pol II and DRB sensitivity-inducing factor (DSIF) are reused for iterative rounds of transcription. Surprisingly, although PAF1 and Spt6 are found throughout the gene body by chromatin immunoprecipitation (ChIP) assays, they show markedly different binding behaviors. Additionally, we found that PAF1 and Spt6 are only recruited after positive transcription elongation factor (P-TEFb)-mediated phosphorylation and RNA Pol II promoter-proximal pause escape. Finally, we observed that PAF1 may be expendable for transcription of highly expressed genes where nucleosome density is low. Thus, our live-cell imaging data provide key constraints to mechanistic models of transcription regulation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Objective: In past work in budding yeast, we identified a nucleosomal region required for proper interactions between the histone chaperone complex yFACT and transcribed genes. Specific histone mutations within this region cause a shift in yFACT occupancy towards the 3' end of genes, a defect that we have attributed to impaired yFACT dissociation from DNA following transcription. In this work we wished to assess the contributions of DNA sequences at the 3' end of genes in promoting yFACT dissociation upon transcription termination., Results: We generated fourteen different alleles of the constitutively expressed yeast gene PMA1, each lacking a distinct DNA fragment across its 3' end, and assessed their effects on occupancy of the yFACT component Spt16. Whereas most of these alleles conferred no defects on Spt16 occupancy, one did cause a modest increase in Spt16 binding at the gene's 3' end. Interestingly, the same allele also caused minor retention of RNA Polymerase II (Pol II) and altered nucleosome occupancy across the same region of the gene. These results suggest that specific DNA sequences at the 3' ends of genes can play roles in promoting efficient yFACT and Pol II dissociation from genes and can also contribute to proper chromatin architecture., (© 2024. The Author(s).)

Background: Molecularly targeted therapies have recently become a hotspot in the treatment of LUAD, with ongoing efforts to identify new effective targets due to individual variability. Among these potential targets, the mitochondrial transcription elongation factor (TEFM) stands out as a crucial molecule involved in mitochondrial synthetic transcriptional processing. Dysregulation of TEFM has been implicated in the development of various diseases; however, its specific role in LUAD remains unclear., Methods: We conducted a comprehensive analysis of TEFM expression in LUAD, leveraging data from the TCGA database. Subsequently, we validated these findings using clinical specimens obtained from the First Affiliated Hospital of Soochow University, employing western blotting and qRT-PCR techniques. Further experimental validation was performed through the transfection of cells with TEFM overexpression, knockdown, and knockout lentiviruses. The effects of TEFM on LUAD were evaluated both in vitro and in vivo using a range of assays, including CCK-8, colony formation, EdU incorporation, Transwell migration, Tunel assay, flow cytometry, JC-1 staining, and xenograft tumour models., Results: Our investigation uncovered that TEFM exhibited elevated expression levels in LUAD and exhibited co-localization with mitochondria. Overexpression of TEFM facilitated malignant processes in LUAD cells, whereas its silencing notably curbed these behaviors and induced mitochondrial depolarization, along with ROS production, culminating in apoptosis. Moreover, the absence of TEFM substantially influenced the expression of mitochondrial transcripts and respiratory chain complexes. Results from nude mouse xenograft tumors further validated that inhibiting TEFM expression markedly hindered tumor growth., Conclusion: TEFM promotes LUAD malignant progression through the EMT pathway and determines apoptosis by affecting the expression of mitochondrial transcripts and respiratory chain complexes, providing a new therapeutic direction for LUAD-targeted therapy., (© 2024. The Author(s).)

Archaeal transcription is carried out by a multi-subunit RNA polymerase (RNAP) that is highly homologous in structure and function to eukaryotic RNAP II. Among the set of basal transcription factors, only Spt5 is found in all domains of life, but Spt5 has been shaped during evolution, which is also reflected in the heterodimerization of Spt5 with Spt4 in Archaea and Eukaryotes. To unravel the mechanistic basis of Spt4/5 function in Archaea, we performed structure-function analyses using the archaeal transcriptional machinery of Pyrococcus furiosus (Pfu). We report single-particle cryo-electron microscopy reconstructions of apo RNAP and the archaeal elongation complex (EC) in the absence and presence of Spt4/5. Surprisingly, Pfu Spt4/5 also binds the RNAP in the absence of nucleic acids in a distinct super-contracted conformation. We show that the RNAP clamp/stalk module exhibits conformational flexibility in the apo state of RNAP and that the enzyme contracts upon EC formation or Spt4/5 engagement. We furthermore identified a contact of the Spt5-NGN domain with the DNA duplex that stabilizes the upstream boundary of the transcription bubble and impacts Spt4/5 activity in vitro. This study, therefore, provides the structural basis for Spt4/5 function in archaeal transcription and reveals a potential role beyond the well-described support of elongation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Facilitates chromatin transcription (FACT) is a histone chaperone that supports transcription through chromatin in vitro, but its functional roles in vivo remain unclear. Here, we analyze the in vivo functions of FACT with the use of multi-omics analysis after rapid FACT depletion from human cells. We show that FACT depletion destabilizes chromatin and leads to transcriptional defects, including defective promoter-proximal pausing and elongation, and increased premature termination of RNA polymerase II. Unexpectedly, our analysis revealed that promoter-proximal pausing depends not only on the negative elongation factor (NELF) but also on the +1 nucleosome, which is maintained by FACT., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Candida albicans, a prominent member of the human microbiome, can make an opportunistic switch from commensal coexistence to pathogenicity accompanied by an epigenetic shift between the white and opaque cell states. This transcriptional switch is under precise regulation by a set of transcription factors (TFs), with Enhanced Filamentous Growth Protein 1 (Efg1) playing a central role. Previous research has emphasized the importance of Efg1's prion-like domain (PrLD) and the protein's ability to undergo phase separation for the white-to-opaque transition of C. albicans. However, the underlying molecular mechanisms of Efg1 phase separation have remained underexplored. In this study, we delved into the biophysical basis of Efg1 phase separation, revealing the significant contribution of both N-terminal (N) and C-terminal (C) PrLDs. Through NMR structural analysis, we found that Efg1 N-PrLD and C-PrLD are mostly disordered but have prominent partial α-helical secondary structures in both domains. NMR titration experiments suggest that the partially helical structures in N-PrLD act as hubs for self-interaction as well as Efg1 interaction with RNA. Using condensed-phase NMR spectroscopy, we uncovered diverse amino acid interactions underlying Efg1 phase separation. Particularly, we highlight the indispensable role of tyrosine residues within the transient α-helical structures of PrLDs particularly in the N-PrLD compared to the C-PrLD in stabilizing phase separation. Our study provides evidence that the transient α-helical structure is present in the phase-separated state and highlights the particular importance of aromatic residues within these structures for phase separation. Together, these results enhance the understanding of C. albicans transcription factor interactions that lead to virulence and provide a crucial foundation for potential antifungal therapies targeting the transcriptional switch., Competing Interests: Declaration of interests N.L.F. is a consultant for Dewpoint Therapeutics and Confluence Therapeutics., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Familial Dysautonomia (FD) is a rare disease caused by ELP1 exon 20 skipping. Here we clarify the role of RNA Polymerase II (RNAPII) and chromatin on this splicing event. A slow RNAPII mutant and chromatin-modifying chemicals that reduce the rate of RNAPII elongation induce exon skipping whereas chemicals that create a more relaxed chromatin exon inclusion. In the brain of a mouse transgenic for the human FD-ELP1 we observed on this gene an age-dependent decrease in the RNAPII density profile that was most pronounced on the alternative exon, a robust increase in the repressive marks H3K27me3 and H3K9me3 and a decrease of H3K27Ac, together with a progressive reduction in ELP1 exon 20 inclusion level. In HEK 293T cells, selective drug-induced demethylation of H3K27 increased RNAPII elongation on ELP1 and SMN2, promoted the inclusion of the corresponding alternative exons, and, by RNA-sequencing analysis, induced changes in several alternative splicing events. These data suggest a co-transcriptional model of splicing regulation in which age-dependent changes in H3K27me3/Ac modify the rate of RNAPII elongation and affect processing of ELP1 alternative exon 20., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Riccardi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Histone 2B ubiquitination (H2Bub) and trimethylation of H3 at lysine 4 (H3K4me3) are associated with transcription activation. However, the function of these modifications in transcription in plants remains largely unknown. Here, we report that coordination of H2Bub and H3K4me3 deposition with the binding of the RNA polymerase-associated factor VERNALIZATION INDEPENDENCE2 (VIP2) to FLOWERING LOCUS C (FLC) modulates flowering time in Arabidopsis. We found that RING domain protein HISTONE MONOUBIQUITINATION1 (HUB1) and HUB2 (we refer as HUB1/2), which are responsible for H2Bub, interact with ARABIDOPSIS TRITHORAX1 (ATX1), which is required for H3K4me3 deposition, to promote the transcription of FLC and repress the flowering time. The atx1-2 hub1-10 hub2-2 triple mutant in FRIGIDIA (FRI) background displayed early flowering like FRI hub1-10 hub2-2 and overexpression of ATX1 failed to rescue the early flowering phenotype of hub1-10 hub2-2. Mutations in HUB1 and HUB2 reduced the ATX1 enrichment at FLC, indicating that HUB1 and HUB2 are required for ATX1 recruitment and H3K4me3 deposition at FLC. We also found that the VIP2 directly binds to HUB1, HUB2, and ATX1 and that loss of VIP2 in FRI hub1-10 hub2-2 and FRI atx1-2 plants resulted in early flowering like that observed in FRI vip2-10. Loss of function of HUB2 and ATX1 impaired VIP2 enrichment at FLC, and reduced the transcription initiation and elongation of FLC. In addition, mutations in VIP2 reduced HUB1 and ATX1 enrichment and H2Bub and H3K4me3 levels at FLC. Together, our findings revealed that HUB1/2, ATX1, and VIP2 coordinately modulate H2Bub and H3K4me3 deposition, FLC transcription, and flowering time., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

In plants, the plant-specific RNA polymerase V (Pol V) transcripts non-coding RNAs and provides a docking platform for the association of accessory proteins in the RNA-directed DNA methylation (RdDM) pathway. Various components have been uncovered that are involved in the process of DNA methylation, but it is still not clear how the transcription of Pol V is regulated. Here, we report that the conserved RNA polymerase II (Pol II) elongator, SPT6L, binds to thousands of intergenic regions in a Pol II-independent manner. The intergenic enrichment of SPT6L, interestingly, co-occupies with the largest subunit of Pol V (NRPE1) and mutation of SPT6L leads to the reduction of DNA methylation but not Pol V enrichment. Furthermore, the association of SPT6L at Pol V loci is dependent on the Pol V associated factor, SPT5L, rather than the presence of Pol V, and the interaction between SPT6L and NRPE1 is compromised in spt5l. Finally, Pol V RIP-seq reveals that SPT6L is required to maintain the amount and length of Pol V transcripts. Our findings thus uncover the critical role of a Pol II conserved elongator in Pol V mediated DNA methylation and transcription, and shed light on the mutual regulation between Pol V and II in plants., (© 2024. The Author(s).)

β-thalassemia is one of the most common monogenic disorders in areas of the tropics and subtropics, which represents a major familial and social burden to local people. The elevated Hb A 2 level, generally specified as greater than 3.5 %, is commonly used as a high efficiency index for screening of β-thalassemia carriers. However, mutations in other genes such as GATA1 and KLF1, could also result in increased Hb A 2 level. In this study, we identified two novel variants in the SUPT5H gene: a frameshift mutation (SUPT5H: c.3032_3033delTG, p.M1011Mfs*9) and a nonsense mutation (SUPT5H: c.397C > T, p.Arg133*) in two Chinese individuals. Utilizing a combination of phenotype analysis, bioinformatics analysis, and functional analysis, we deduced that these two variants modified the SUPT5H protein's structure, thereby impacting its function and consequently leading to the heightened Hb A 2 level phenotype found in the carriers. Furthermore, through a comprehensive literature review, a mutation spectrum was consolidated for SUPT5H, an investigation into the genotype-phenotype correlation was conducted, and factors known to influence Hb A 2 levels were identified. Based on this in-depth understanding, clinicians are better equipped to carry out large scale screenings in regions with high prevalence of β-thalassemia., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The RNA polymerase II (Pol II) elongation rate influences poly(A) site selection, with slow and fast Pol II derivatives causing upstream and downstream shifts, respectively, in poly(A) site utilization. In yeast, depletion of either of the histone chaperones FACT or Spt6 causes an upstream shift of poly(A) site use that strongly resembles the poly(A) profiles of slow Pol II mutant strains. Like slow Pol II mutant strains, FACT- and Spt6-depleted cells exhibit Pol II processivity defects, indicating that both Spt6 and FACT stimulate the Pol II elongation rate. Poly(A) profiles of some genes show atypical downstream shifts; this subset of genes overlaps well for FACT- or Spt6-depleted strains but is different from the atypical genes in Pol II speed mutant strains. In contrast, depletion of histone H3 or H4 causes a downstream shift of poly(A) sites for most genes, indicating that nucleosomes inhibit the Pol II elongation rate in vivo. Thus, chromatin-based control of the Pol II elongation rate is a potential mechanism, distinct from direct effects on the cleavage/polyadenylation machinery, to regulate alternative polyadenylation in response to genetic or environmental changes., Competing Interests: Competing interests statement:The authors declare no competing interest.

Mechanisms of functional cross-talk between global transcriptional repression and efficient DNA damage repair during genotoxic stress are poorly known. In this study, using human AF9 as representative of Super Elongation Complex (SEC) components, we delineate detailed mechanisms of these processes. Mechanistically, we describe that Poly-Serine domain-mediated oligomerization is pre-requisite for AF9 YEATS domain-mediated TFIID interaction-dependent SEC recruitment at the promoter-proximal region for release of paused RNA polymerase II. Interestingly, during genotoxic stress, CaMKII-mediated phosphorylation-dependent nuclear export of AF9-specific deacetylase HDAC5 enhances concomitant PCAF-mediated acetylation of K339 residue. This causes monomerization of AF9 and reduces TFIID interaction for transcriptional downregulation. Furthermore, the K339 acetylation-dependent enhanced AF9-DNA-PKc interaction leads to phosphorylation at S395 residue which reduces AF9-SEC interaction resulting in transcriptional downregulation and efficient repair of DNA damage. After repair, nuclear re-entry of HDAC5 reduces AF9 acetylation and restores its TFIID and SEC interaction to restart transcription., (© 2024. The Author(s).)

In cancer therapy, DNA intercalators are mainly known for their capacity to kill cells by inducing DNA damage. Recently, several DNA intercalators have attracted much interest given their ability to inhibit RNA Polymerase I transcription (BMH-21), evict histones (Aclarubicin) or induce chromatin trapping of FACT (Curaxin CBL0137). Interestingly, these DNA intercalators lack the capacity to induce DNA damage while still retaining cytotoxic effects and stabilize p53. Herein, we report that these DNA intercalators impact chromatin biology by interfering with the chromatin stability of RNA polymerases I, II and III. These three compounds have the capacity to induce degradation of RNA polymerase II and they simultaneously enable the trapping of Topoisomerases TOP2A and TOP2B on the chromatin. In addition, BMH-21 also acts as a catalytic inhibitor of Topoisomerase II, resembling Aclarubicin. Moreover, BMH-21 induces chromatin trapping of the histone chaperone FACT and propels accumulation of Z-DNA and histone eviction, similarly to Aclarubicin and CBL0137. These DNA intercalators have a cumulative impact on general transcription machinery by inducing accumulation of topological defects and impacting nuclear chromatin. Therefore, their cytotoxic capabilities may be the result of compounding deleterious effects on chromatin homeostasis., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Transcriptional elongation by RNA polymerase II (RNAPII) through chromatin is a dynamic and highly regulated step of eukaryotic gene expression. A combination of transcript elongation factors (TEFs) including modulators of RNAPII activity and histone chaperones facilitate efficient transcription on nucleosomal templates. Biochemical and genetic analyses, primarily performed in Arabidopsis, provided insight into the contribution of TEFs to establish gene expression patterns during plant growth and development. In addition to summarising the role of TEFs in plant gene expression, we emphasise in our review recent advances in the field. Thus, mechanisms are presented how aberrant intragenic transcript initiation is suppressed by repressing transcriptional start sites within coding sequences. We also discuss how transcriptional interference of ongoing transcription with neighbouring genes is prevented. Moreover, it appears that plants make no use of promoter-proximal RNAPII pausing in the way mammals do, but there are nucleosome-defined mechanism(s) that determine the efficiency of mRNA synthesis by RNAPII. Accordingly, a still growing number of processes related to plant growth, development and responses to changing environmental conditions prove to be regulated at the level of transcriptional elongation., (© 2023 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Platinum resistance remains a major contributor to the poor prognosis of ovarian cancer. Anti-apoptotic protein myeloid cell leukemia-1 (MCL-1) has emerged as a promising target for overcoming drug resistance, but different cancer cells utilize distinct protein degradation pathways to alter MCL-1 level. We systematically investigated E3 ligases to identify novel candidates that mediate platinum resistance in ovarian cancer. Transcription Elongation Factor B (TCEB3) has been identified as a novel E3 ligase recognition subunit that targets MCL-1 in the cytoplasm during platinum treatment other than its traditional function of targeting the Pol II in the nuclear compartment. TCEB3 expression is downregulated in platinum-resistant cell lines and this low expression is associated with poor prognosis. The ubiquitination of MCL-1 induced by TCEB3 leads to cell death in ovarian cancer. Moreover, platinum treatment increased the cytoplasm proportion of TCEB3, and the cytoplasm localization of TCEB3 is important for its targeting of MCL-1. This study emphasizes the dual function of TCEB3 in homeostasis maintenance and in cell fate determination under different conditions, and provides a new insight into drug resistance in ovarian cancer., (© 2024 Federation of American Societies for Experimental Biology.)

Background: Splicing factors are vital for the regulation of RNA splicing, but some have also been implicated in regulating transcription. The underlying molecular mechanisms of their involvement in transcriptional processes remain poorly understood., Results: Here, we describe a direct role of splicing factor RBM22 in coordinating multiple steps of RNA Polymerase II (RNAPII) transcription in human cells. The RBM22 protein widely occupies the RNAPII-transcribed gene locus in the nucleus. Loss of RBM22 promotes RNAPII pause release, reduces elongation velocity, and provokes transcriptional readthrough genome-wide, coupled with production of transcripts containing sequences from downstream of the gene. RBM22 preferentially binds to the hyperphosphorylated, transcriptionally engaged RNAPII and coordinates its dynamics by regulating the homeostasis of the 7SK-P-TEFb complex and the association between RNAPII and SPT5 at the chromatin level., Conclusions: Our results uncover the multifaceted role of RBM22 in orchestrating the transcriptional program of RNAPII and provide evidence implicating a splicing factor in both RNAPII elongation kinetics and termination control., (© 2024. The Author(s).)

Background: Through whole-exome sequencing of 60 formalin-fixed paraffin-embedded Nigerian (NGRn) benign prostatic hyperplasia (BPH) samples, we identified germline and somatic alterations in apoptotic pathways impacting BPH development and progression. Prostate enlargement is a common occurrence in male aging; however, this enlargement can lead to lower urinary tract symptoms that negatively impact quality of life. This impact is disproportionately present in men of African ancestry. BPH pathophysiology is poorly understood and studies examining non-European populations are lacking., Methods: In this study, NGRn BPH, normal prostate, and prostate cancer (PCa) tumor samples were sequenced and compared to characterize genetic alterations in NGRn BPH., Results: Two hundred and two nonbenign, ClinVar-annotated germline variants were present in NGRn BPH samples. Six genes [BRCA1 (92%), HSD3B1 (85%), TP53 (37%), PMS2 (23%), BARD1 (20%), and BRCA2 (17%)] were altered in at least 10% of samples; however, compared to NGRn normal and tumor, the frequency of alterations in BPH samples showed no significant differences at the gene or variant level. BRCA2&#95;rs11571831 and TP53&#95;rs1042522 germline alterations had a statistically significant co-occurrence interaction in BPH samples. In at least two BPH samples, 173 genes harbored somatic variants known to be clinically actionable. Three genes (COL18A1, KIF16B, and LRP1) showed a statistically significant (p < 0.05) higher frequency in BPH. NGRn BPH also had five gene pairs (PKD1/KIAA0100, PKHD1/PKD1, DNAH9/LRP1B, NWD1/DCHS2, and TCERG1/LMTK2) with statistically significant co-occurring interactions. Two hundred and seventy-nine genes contained novel somatic variants in NGRn BPH. Three genes (CABP1, FKBP1C, and RP11-595B24.2) had a statistically significant (p < 0.05) higher alteration frequency in NGRn BPH and three were significantly higher in NGRn tumor (CACNA1A, DMKN, and CACNA2D2). Pairwise Fisher's exact tests showed 14 gene pairs with statistically significant (p < 0.05) interactions and four interactions approaching significance (p < 0.10). Mutational patterns in NGRn BPH were similar to COSMIC (Catalog of Somatic Mutations in Cancer) signatures associated with aging and dysfunctional DNA damage repair., Conclusions: NGRn BPH contained significant germline alteration interactions (BRCA2&#95;rs11571831 and TP53&#95;rs1042522) and increased somatic alteration frequencies (LMTK2, LRP1, COL18A1, CABP1, and FKBP1C) that impact apoptosis. Normal prostate development is maintained by balancing apoptotic and proliferative activity. Dysfunction in either mechanism can lead to abnormal prostate growth. This work is the first to examine genomic sequencing in NGRn BPH and provides data that fill known gaps in the understanding BPH and how it impacts men of African ancestry., (© 2024 Wiley Periodicals LLC.)

Leukaemia of various subtypes are driven by distinct chromosomal rearrangement or genetic abnormalities. The leukaemogenic fusion transcripts or genetic mutations serve as molecular markers for minimal residual disease (MRD) monitoring. The current study evaluated the applicability of several droplet digital PCR assays for the detection of these targets at RNA and DNA levels (atypical BCR::ABL1 e19a2, e23a2ins52, e13a2ins74, rare types of CBFB::MYH11 (G and I), PCM1::JAK2, KMT2A::ELL2, PICALM::MLLT10 fusion transcripts and CEBPA frame-shift and insertion/duplication mutations) with high sensitivity. The analytical performances were assessed by the limit of blanks, limit of detection, limit of quantification and linear regression. Our data demonstrated serial MRD monitoring for patients at molecular level could become "digitalized", which was deemed important to guide clinicians in treatment decision for better patient care., (© 2024. The Author(s).)

The B complex is a key intermediate stage of spliceosome assembly. To improve the structural resolution of monomeric, human spliceosomal B (hB) complexes and thereby generate a more comprehensive hB molecular model, we determined the cryo-EM structure of B complex dimers formed in the presence of ATP γ S. The enhanced resolution of these complexes allows a finer molecular dissection of how the 5' splice site (5'ss) is recognized in hB, and new insights into molecular interactions of FBP21, SNU23 and PRP38 with the U6/5'ss helix and with each other. It also reveals that SMU1 and RED are present as a heterotetrameric complex and are located at the interface of the B dimer protomers. We further show that MFAP1 and UBL5 form a 5' exon binding channel in hB, and elucidate the molecular contacts stabilizing the 5' exon at this stage. Our studies thus yield more accurate models of protein and RNA components of hB complexes. They further allow the localization of additional proteins and protein domains (such as SF3B6, BUD31 and TCERG1) whose position was not previously known, thereby uncovering new functions for B-specific and other hB proteins during pre-mRNA splicing., (© 2024. The Author(s).)

Mutations in APC, found in 80% of colon caner, enhance β-catenin stabilization, which is the initial step of colonic tumorigenesis. However, the core transcriptional mechanism underlying the induction of colon cancer stemness by stable β-catenin remains unclear. Here, we found that inducible inhibition of β-catenin suppressed elongation of Pol II and RNA polymerase-associated factor 1 complex (PAF1C) around the transcription start site (TSS) of LGR5. Moreover, stable β-catenin enhanced the formation of active Pol II complex cooperatively with CDC73 and CDK9 by facilitating the recruitment of DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF) complexes to the Pol II complex. Subsequently, stable β-catenin facilitated the formation of the Pol II-DSIF-PAF1C complex, suggesting that stable β-catenin induces cancer stemness by stimulating active Pol II complex through NELF and PAF1C. Furthermore, NELF or PAF1C inhibition recapitulated the changes in cancer stemness-related gene expression induced by the inhibition of stable β-catenin and suppressed colon cancer stemness. Additionally, the chemical inhibition of CDK12 (a downstream transcription CDK of PAF1C) suppressed colon cancer stemness. These results suggest that NELF and PAF1C are the core transcriptional machineries that control expression of colon cancer stemness-inducing genes and may be therapeutic targets for colon cancer., (© 2024. The Author(s).)

RSC (remodels the structure of chromatin) is an essential ATP-dependent chromatin remodeling complex in Saccharomyces cerevisiae. RSC utilizes its ATPase subunit, Sth1, to slide or remove nucleosomes. RSC has been shown to regulate the width of the nucleosome-depleted regions (NDRs) by sliding the flanking nucleosomes away from NDRs. As such, when RSC is depleted, nucleosomes encroach NDRs, leading to transcription initiation defects. In this study, we examined the effects of the catalytic-dead Sth1 on transcription and compared them to those observed during acute and rapid Sth1 depletion by auxin-induced degron strategy. We found that rapid depletion of Sth1 reduces recruitment of TBP and Pol II in highly transcribed genes, as would be expected considering its role in regulating chromatin structure at promoters. In contrast, cells harboring the catalytic-dead Sth1 (sth1-K501R) exhibited a severe reduction in TBP binding, but, surprisingly, also displayed a substantial accumulation in Pol II occupancies within coding regions. The Pol II occupancies further increased upon depleting endogenous Sth1 in the catalytic-dead mutant, suggesting that the inactive Sth1 contributes to Pol II accumulation in coding regions. Notwithstanding the Pol II increase, the ORF occupancies of histone chaperones, FACT and Spt6 were significantly reduced in the mutant. These results suggest a potential role for RSC in recruiting/retaining these chaperones in coding regions. Pol II accumulation despite substantial reductions in TBP, FACT, and Spt6 occupancies in the catalytic-dead mutant could indicate severe transcription elongation and termination defects. Such defects would be consistent with studies showing that RSC is recruited to coding regions in a transcription-dependent manner. Thus, these findings imply a role for RSC in transcription elongation and termination processes, in addition to its established role in transcription initiation., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Chhabi Govind reports financial support was provided by National Institutes of Health. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The super elongation complex (SEC) containing positive transcription elongation factor b plays a critical role in regulating transcription elongation. AFF1 and AFF4, two members of the AF4/FMR2 family, act as central scaffold proteins of SEC and are associated with various human diseases. However, their precise roles in transcriptional control remain unclear. Here, we investigate differences in the genomic distribution patterns of AFF1 and AFF4 around transcription start sites (TSSs). AFF1 mainly binds upstream of the TSS, while AFF4 is enriched downstream of the TSS. Notably, disruption of AFF4 results in slow elongation and early termination in a subset of AFF4-bound active genes, whereas AFF1 deletion leads to fast elongation and transcriptional readthrough in the same subset of genes. Additionally, AFF1 knockdown increases AFF4 levels at chromatin, and vice versa. In summary, these findings demonstrate that AFF1 and AFF4 function antagonistically to regulate RNA polymerase II transcription., (© The Author(s) (2023). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, CEMCS, CAS.)

A t(4;11) leukemia model established from CRISPR-engineered chromosomal translocations between the KMT2A and AFF1 genes recapitulate proteomic, epigenomic, and transcriptomic features of primary patient leukemias.

Background: Post-transcriptional regulation of mRNA induced by microRNA is known crucial in tumor occurrence, progression, and metastasis. This study aims at identifying significant miRNA-mRNA axes for stomach adenocarcinomas (STAD)., Method: RNA expression profiles were collected from The Cancer Genome Atlas (TCGA) and GEO database for screening differently expressed RNAs and miRNAs (DE-miRNAs/DE-mRNAs). Functional enrichment analysis was conducted with Hiplot and DAVID-mirPath. Connectivity MAP was applied in compounds prediction. MiRNA-mRNA axes were forecasted by TarBase and MiRTarBase. Real-time reverse transcription polymerase chain reaction (RT-qPCR) of stomach specimen verified these miRNA-mRNA pairs. Diagnosis efficacy of miRNA-mRNA interactions was measured by Receiver operation characteristic curve and Decision Curve Analysis. Clinical and survival analysis were also carried out. CIBERSORT and ESTIMATE was employed for immune microenvironment measurement., Result: Totally 228 DE-mRNAs (105 upregulated and 123 downregulated) and 38 DE-miRNAs (22 upregulated and 16 downregulated) were considered significant. TarBase and MiRTarBase identified 18 miRNA-mRNA pairs, 12 of which were verified in RT-qPCR. The network of miR-301a-3p/ELL2 and miR-1-3p/ANXA2 were established and verified in external validation. The model containing all 4 signatures showed better diagnosis ability. Via interacting with M0 macrophage and resting mast cell, these miRNA-mRNA axes may influence tumor microenvironment., Conclusion: This study established a miRNA-mRNA network via bioinformatic analysis and experiment validation for STAD.

The elongation stage of transcription by RNA polymerase II (RNA Pol II) is central to the regulation of gene expression in response to developmental and environmental cues in metazoan. Dysregulated transcriptional elongation has been associated with developmental defects as well as disease and aging processes. Decades of genetic and biochemical studies have painstakingly identified and characterized an ensemble of factors that regulate RNA Pol II elongation. This review summarizes recent findings taking advantage of genetic engineering techniques that probe functions of elongation factors in vivo. We propose a revised model of elongation control in this accelerating field by reconciling contradictory results from the earlier biochemical evidence and the recent in vivo studies. We discuss how elongation factors regulate promoter-proximal RNA Pol II pause release, transcriptional elongation rate and processivity, RNA Pol II stability and RNA processing, and how perturbation of these processes is associated with developmental disorders, neurodegenerative disease, cancer, and aging., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023. Published by Elsevier Inc.)

Heterochromatin is a condensed chromatin structure that represses transcription of repetitive DNA elements and developmental genes, and is required for genome stability. Paradoxically, transcription of heterochromatic sequences is required for establishment of heterochromatin in diverse eukaryotic species. As such, components of the transcriptional machinery can play important roles in establishing heterochromatin. How these factors coordinate with heterochromatin proteins at nascent heterochromatic transcripts remains poorly understood. In the model eukaryote Schizosaccharomyces pombe (S. pombe), heterochromatin nucleation can be coupled to processing of nascent transcripts by the RNA interference (RNAi) pathway, or to other post-transcriptional mechanisms that are RNAi-independent. Here we show that the RNA polymerase II processivity factor Spt5 negatively regulates heterochromatin in S. pombe through its C-terminal domain (CTD). The Spt5 CTD is analogous to the CTD of the RNA polymerase II large subunit, and is comprised of multiple repeats of an amino acid motif that is phosphorylated by Cdk9. We provide evidence that genetic ablation of Spt5 CTD phosphorylation results in aberrant RNAi-dependent nucleation of heterochromatin at an ectopic location, as well as inappropriate spread of heterochromatin proximal to centromeres. In contrast, truncation of Spt5 CTD repeat number enhanced RNAi-independent heterochromatin formation and bypassed the requirement for RNAi. We relate these phenotypes to the known Spt5 CTD-binding factor Prf1/Rtf1. This separation of function argues that Spt5 CTD phosphorylation and CTD length restrict heterochromatin through unique mechanisms. More broadly, our findings argue that length and phosphorylation of the Spt5 CTD repeat array have distinct regulatory effects on transcription., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 MacKinnon et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Transcription elongation requires elaborate coordination between the transcriptional machinery and chromatin regulatory factors to successfully produce RNA while preserving the epigenetic landscape. Recent structural and genomic studies have highlighted that suppressor of Ty 6 (Spt6), a conserved histone chaperone and transcription elongation factor, sits at the crux of the transcription elongation process. Other recent studies have revealed that Spt6 also promotes DNA replication and genome integrity. Here, we review recent studies of Spt6 that have provided new insights into the mechanisms by which Spt6 controls transcription and have revealed the breadth of Spt6 functions in eukaryotic cells., Competing Interests: Declaration of interests The authors declare no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

We identified a novel mutation in the SUPT5H gene in a Chinese female who presented with a β-thalassemia trait. The substitution of c.193C > T (p.Arg65*) leads to a premature stop codon on residue 65 and could be associated with haploinsufficiency. This variant was inherited from the mother who also had the asymptomatic phenotype of β-thalassemia trait. Our case further supports the role of SUPT5H as a potential β-globin chain production-modulating gene.

Familial dysautonomia (FD) is a rare neurodevelopmental and neurodegenerative disease caused by a splicing mutation in the Elongator Acetyltransferase Complex Subunit 1 (ELP1) gene. The reduction in ELP1 mRNA and protein leads to the death of retinal ganglion cells (RGCs) and visual impairment in all FD patients. Currently patient symptoms are managed, but there is no treatment for the disease. We sought to test the hypothesis that restoring levels of Elp1 would thwart the death of RGCs in FD. To this end, we tested the effectiveness of two therapeutic strategies for rescuing RGCs. Here we provide proof-of-concept data that gene replacement therapy and small molecule splicing modifiers effectively reduce the death of RGCs in mouse models for FD and provide pre-clinical foundational data for translation to FD patients., (© 2023. The Author(s).)

Controlled release of promoter-proximal paused RNA polymerase II (RNA Pol II) is crucial for gene regulation. However, studying RNA Pol II pausing is challenging, as pause-release factors are almost all essential. In this study, we identified heterozygous loss-of-function mutations in SUPT5H, which encodes SPT5, in individuals with β-thalassemia. During erythropoiesis in healthy human cells, cell cycle genes were highly paused as cells transition from progenitors to precursors. When the pathogenic mutations were recapitulated by SUPT5H editing, RNA Pol II pause release was globally disrupted, and as cells began transitioning from progenitors to precursors, differentiation was delayed, accompanied by a transient lag in erythroid-specific gene expression and cell cycle kinetics. Despite this delay, cells terminally differentiate, and cell cycle phase distributions normalize. Therefore, hindering pause release perturbs proliferation and differentiation dynamics at a key transition during erythropoiesis, identifying a role for RNA Pol II pausing in temporally coordinating the cell cycle and erythroid differentiation., Competing Interests: Declaration of interests V.G.S. serves as an advisor to and/or has equity in Branch Biosciences, Ensoma, Novartis, Forma, Sana Biotechnology, and Cellarity, all unrelated to the present work. J.C.U. is an employee of Illumina, Inc., unrelated to the present work. R.I. is a founder, board member, and shareholder of Cellforma, unrelated to the present work., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Suppressor of Ty homolog-5 (SPT5) discovered in the yeast mutant screens as a suppressor of mutation caused by the insertion of the Transposons of yeast (Ty) element along with SPT4, with which it forms a holoenzyme complex known as DRB sensitivity-inducing factor (DSIF) and plays an essential role in the regulation of transcription. SPT5 is a highly conserved protein across all three domains of life and performs critical functions in transcription, starting from promoter-proximal pausing to termination. We also highlight the emerging role of SPT5 in other non-canonical functions, such as the regulation of post-translational modifications (PTM) and the transcriptional regulation of non-coding genes. Also, in brief, we highlight the clinical implications of SPT5 dysregulation., Competing Interests: No conflicts of interest exist, according to the authors., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Cornelia de Lange Syndrome (CdLS) is a rare, dominantly inherited multisystem developmental disorder characterized by highly variable manifestations of growth and developmental delays, upper limb involvement, hypertrichosis, cardiac, gastrointestinal, craniofacial, and other systemic features. Pathogenic variants in genes encoding cohesin complex structural subunits and regulatory proteins (NIPBL, SMC1A, SMC3, HDAC8, and RAD21) are the major pathogenic contributors to CdLS. Heterozygous or hemizygous variants in the genes encoding these five proteins have been found to be contributory to CdLS, with variants in NIPBL accounting for the majority (>60%) of cases, and the only gene identified to date that results in the severe or classic form of CdLS when mutated. Pathogenic variants in cohesin genes other than NIPBL tend to result in a less severe phenotype. Causative variants in additional genes, such as ANKRD11, EP300, AFF4, TAF1, and BRD4, can cause a CdLS-like phenotype. The common role that these genes, and others, play as critical regulators of developmental transcriptional control has led to the conditions they cause being referred to as disorders of transcriptional regulation (or "DTRs"). Here, we report the results of a comprehensive molecular analysis in a cohort of 716 probands with typical and atypical CdLS in order to delineate the genetic contribution of causative variants in cohesin complex genes as well as novel candidate genes, genotype-phenotype correlations, and the utility of genome sequencing in understanding the mutational landscape in this population., (© 2023 Wiley Periodicals LLC.)

Background: Neurodevelopmental disorders (NDDs) are heterogeneous, debilitating conditions that include motor and cognitive disability and social deficits. The genetic factors underlying the complex phenotype of NDDs remain to be elucidated. Accumulating evidence suggest that the Elongator complex plays a role in NDDs, given that patient-derived mutations in its ELP2, ELP3, ELP4 and ELP6 subunits have been associated with these disorders. Pathogenic variants in its largest subunit ELP1 have been previously found in familial dysautonomia and medulloblastoma, with no link to NDDs affecting primarily the central nervous system., Methods: Clinical investigation included patient history and physical, neurological and magnetic resonance imaging (MRI) examination. A novel homozygous likely pathogenic ELP1 variant was identified by whole-genome sequencing. Functional studies included in silico analysis of the mutated ELP1 in the context of the holo-complex, production and purification of the ELP1 harbouring the identified mutation and in vitro analyses using microscale thermophoresis for tRNA binding assay and acetyl-CoA hydrolysis assay. Patient fibroblasts were harvested for tRNA modification analysis using HPLC coupled to mass spectrometry., Results: We report a novel missense mutation in the ELP1 identified in two siblings with intellectual disability and global developmental delay. We show that the mutation perturbs the ability of ELP123 to bind tRNAs and compromises the function of the Elongator in vitro and in human cells., Conclusion: Our study expands the mutational spectrum of ELP1 and its association with different neurodevelopmental conditions and provides a specific target for genetic counselling., (© 2023. Crown.)

The Paf1 complex (Paf1C) is a conserved transcription elongation factor that regulates transcription elongation efficiency, facilitates co-transcriptional histone modifications, and impacts molecular processes linked to RNA synthesis, such as polyA site selection. Coupling of the activities of Paf1C to transcription elongation requires its association with RNA polymerase II (Pol II). Mutational studies in yeast identified Paf1C subunits Cdc73 and Rtf1 as important mediators of Paf1C association with Pol II on active genes. While the interaction between Rtf1 and the general elongation factor Spt5 is relatively well-understood, the interactions involving Cdc73 have not been fully elucidated. Using a site-specific protein cross-linking strategy in yeast cells, we identified direct interactions between Cdc73 and two components of the Pol II elongation complex, the elongation factor Spt6 and the largest subunit of Pol II. Both of these interactions require the tandem SH2 domain of Spt6. We also show that Cdc73 and Spt6 can interact in vitro and that rapid depletion of Spt6 dissociates Paf1 from chromatin, altering patterns of Paf1C-dependent histone modifications genome-wide. These results reveal interactions between Cdc73 and the Pol II elongation complex and identify Spt6 as a key factor contributing to the occupancy of Paf1C at active genes in Saccharomyces cerevisiae., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Objective: To explore the correlation of polymorphisms of AF4/FMR2 family genes and IL-10 gene with genetic susceptibility to ankylosing spondylitis (AS) and identify the high-risk factors of AS., Methods: This case-control study was conducted among 207 AS patients and 321 healthy individuals. The tag single nucleotide polymorphisms (SNPs) rs340630, rs241084, rs10865035, rs1698105, and rs1800896 of the AF4/FMR2 family gene and IL-10 gene of the AS patients were genotyped, and the distribution frequencies of the genotypes and alleles were analyzed to explore the relationship between different genetic models and AS and the gene-gene and gene-environment interactions., Results: Gender ratio, smoking history, drinking history, hypertension, erythrocyte sedimentation rate and C-reactive protein differed significantly between the case group and the control group ( P < 0.05). The dominant model and recessive model of AFF1 rs340630, the recessive model of AFF3 rs10865035, and the recessive model of IL-10 rs1800896 were significantly different between the two groups ( P =0.031, 0.010, 0.031, and 0.019, respectively). Gene-environment interaction analysis suggested that the interaction model incorporating AFF1 rs340630, AFF2 rs241084, AFF3 rs10865035, AFF4 rs1698105, IL-10 rs1800896, smoking history and drinking history was the best model. The genes related with AF4/FMR2 and IL-10 were enriched in the biological processes of AF4 super extension complex, interleukin family signal transduction, cytokine stimulation and apoptosis. The expression levels of AF4/FMR2 and IL-10 were positively correlated with immune infiltration ( r > 0)., Conclusion: The SNPs of AF4/FMR2 and IL-10 genes are associated with the susceptibility to AS, and the interactions of AF4/FMR2 and IL-10 genes with the environmental factors contributes causes AS through immune infiltration.

Transcription is highly regulated by a variety of transcription factors, among which NusA and NusG act contradictorily in Escherichia coli (E. coli) that NusA stabilizes a paused RNA polymerase (RNAP) and NusG suppresses it. The mechanism of the NusA and NusG regulations on RNAP transcription has been addressed, but their effect on the conformational changes of the transcription bubble correlated with transcription kinetics remains elusive. By using single-molecule magnetic trap, we identify a reduction in the transcription rate of ∼40% events by NusA. Although the rest ∼60% of transcription events exhibit unaffected transcription rates, a NusA-enhanced standard deviation of the transcription rate is observed. NusA remodeling also increases the extent of DNA unwinding in the transcription bubble by 1-2 base pairs, which can be reduced by NusG. The NusG remodeling is more significant on the RNAP molecules with reduced transcription rates rather than those without. Our results provide a quantitative view on the mechanisms of transcriptional regulation by NusA and NusG factors.