Human promoter directionality is determined by transcriptional initiation and the opposing activities of INTS11 and CDK9.

RNA polymerase II (RNAPII) transcription initiates bidirectionally at many human protein-coding genes. Sense transcription usually dominates and leads to messenger RNA production, whereas antisense transcription rapidly terminates. The basis for this directionality is not fully understood. Here, we show that sense transcriptional initiation is more efficient than in the antisense direction, which establishes initial promoter directionality. After transcription begins, the opposing functions of the endonucleolytic subunit of Integrator, INTS11, and cyclin-dependent kinase 9 (CDK9) maintain directionality. Specifically, INTS11 terminates antisense transcription, whereas sense transcription is protected from INTS11-dependent attenuation by CDK9 activity. Strikingly, INTS11 attenuates transcription in both directions upon CDK9 inhibition, and the engineered recruitment of CDK9 desensitises transcription to INTS11. Therefore, the preferential initiation of sense transcription and the opposing activities of CDK9 and INTS11 explain mammalian promoter directionality.

Age-dependent regulation of ELP1 exon 20 splicing in Familial Dysautonomia by RNA Polymerase II kinetics and chromatin structure.

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.

FACT maintains chromatin architecture and thereby stimulates RNA polymerase II pausing during transcription in vivo.

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.

Post-translational modification-dependent oligomerization switch in regulation of global transcription and DNA damage repair during genotoxic stress.

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.

Chromatin regulates alternative polyadenylation via the RNA polymerase II elongation rate.

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.

A conserved Pol II elongator SPT6L mediates Pol V transcription to regulate RNA-directed DNA methylation in Arabidopsis.

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.

Structural basis of archaeal RNA polymerase transcription elongation and Spt4/5 recruitment.

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.

Key interactions of RNA polymerase with 6S RNA and secondary channel factors during pRNA synthesis.

Small non-coding 6S RNA mimics DNA promoters and binds to the σ70 holoenzyme of bacterial RNA polymerase (RNAP) to suppress transcription of various genes mainly during the stationary phase of cell growth or starvation. This inhibition can be relieved upon synthesis of short product RNA (pRNA) performed by RNAP from the 6S RNA template. Here, we have shown that pRNA synthesis depends on specific contacts of 6S RNA with RNAP and interactions of the σ finger with the RNA template in the active site of RNAP, and is also modulated by the secondary channel factors. We have adapted a molecular beacon assay with fluorescently labeled σ70 to analyze 6S RNA release during pRNA synthesis. We found the kinetics of 6S RNA release to be oppositely affected by mutations in the σ finger and in the CRE pocket of core RNAP, similarly to the reported role of these regions in promoter-dependent transcription. Secondary channel factors, DksA and GreB, inhibit pRNA synthesis and 6S RNA release from RNAP, suggesting that they may contribute to the 6S RNA-mediated switch in transcription during stringent response. Our results demonstrate that pRNA synthesis depends on a similar set of contacts between RNAP and 6S RNA as in the case of promoter-dependent transcription initiation and reveal that both processes can be regulated by universal transcription factors acting on RNAP.

Structural basis of Integrator-dependent RNA polymerase II termination.

The Integrator complex can terminate RNA polymerase II (Pol II) in the promoter-proximal region of genes. Previous work has shed light on how Integrator binds to the paused elongation complex consisting of Pol II, the DRB sensitivity-inducing factor (DSIF) and the negative elongation factor (NELF) and how it cleaves the nascent RNA transcript1, but has not explained how Integrator removes Pol II from the DNA template. Here we present three cryo-electron microscopy structures of the complete Integrator-PP2A complex in different functional states. The structure of the pre-termination complex reveals a previously unresolved, scorpion-tail-shaped INTS10-INTS13-INTS14-INTS15 module that may use its 'sting' to open the DSIF DNA clamp and facilitate termination. The structure of the post-termination complex shows that the previously unresolved subunit INTS3 and associated sensor of single-stranded DNA complex (SOSS) factors prevent Pol II rebinding to Integrator after termination. The structure of the free Integrator-PP2A complex in an inactive closed conformation2 reveals that INTS6 blocks the PP2A phosphatase active site. These results lead to a model for how Integrator terminates Pol II transcription in three steps that involve major rearrangements.

RBM22 regulates RNA polymerase II 5' pausing, elongation rate, and termination by coordinating 7SK-P-TEFb complex and SPT5.

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.

TCEB3 initiates ovarian cancer apoptosis by mediating ubiquitination and degradation of MCL-1.

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.

ATX1 and HUB1/2 promote recruitment of the transcription elongation factor VIP2 to modulate the floral transition in Arabidopsis.

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.

Application of droplet digital PCR in minimal residual disease monitoring of rare fusion transcripts and mutations in haematological malignancies.

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.

Structural basis of exoribonuclease-mediated mRNA transcription termination.

Efficient termination is required for robust gene transcription. Eukaryotic organisms use a conserved exoribonuclease-mediated mechanism to terminate the mRNA transcription by RNA polymerase II (Pol II)1-5. Here we report two cryogenic electron microscopy structures of Saccharomyces cerevisiae Pol II pre-termination transcription complexes bound to the 5'-to-3' exoribonuclease Rat1 and its partner Rai1. Our structures show that Rat1 displaces the elongation factor Spt5 to dock at the Pol II stalk domain. Rat1 shields the RNA exit channel of Pol II, guides the nascent RNA towards its active centre and stacks three nucleotides at the 5' terminus of the nascent RNA. The structures further show that Rat1 rotates towards Pol II as it shortens RNA. Our results provide the structural mechanism for the Rat1-mediated termination of mRNA transcription by Pol II in yeast and the exoribonuclease-mediated termination of mRNA transcription in other eukaryotes.

Structure and interactions of prion-like domains in transcription factor Efg1 phase separation.

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.

Chromatin damage generated by DNA intercalators leads to degradation of RNA Polymerase II.

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.

SUPT5H mutations associated with elevation of Hb A2 level: Identification of two novel variants and literature review.

ß-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 A2 level, generally specified as greater than 3.5 %, is commonly used as a high efficiency index for screening of ß-thalassemia carriers. However, mutations in other genes such as GATA1 and KLF1, could also result in increased Hb A2 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 A2 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 A2 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 ß-thalassemia.

Cryo-EM analyses of dimerized spliceosomes provide new insights into the functions of B complex proteins.

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.

Whole-exome sequencing of Nigerian benign prostatic hyperplasia reveals increased alterations in apoptotic pathways.

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_rs11571831 and TP53_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_rs11571831 and TP53_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.