A (GCC) Repeat in the Untranslated Region of Human SBF1 Departs from Hardy-Weinberg Equilibrium in Human and Links to Late-onset Neurocognitive Disorder.

The human SBF1 (SET binding factor 1) gene, alternatively known as MTMR5, is predominantly expressed in the brain, and its epigenetic dysregulation is linked to late-onset neurocognitive disorders (NCDs), such as Alzheimer’s disease. This gene contains a (GCC)-repeat at the interval between +1 and +60 of the transcription start site (SBF1-202 ENST00000380817.8). Sequencing of the SBF1 (GCC)-repeat in a sample of 542 Iranian individuals, consisting of late-onset NCDs (N=260) and controls (N=282) revealed a predominantly bi-allelic locus for this STR, consisting of 8 and 9 repeats, with allele frequencies ranging from 0.39 to 0.55, and four other alleles with frequencies of <0.03 across the two groups. Overall heterozygosity for the observed alleles was significantly less than expected in the NCD and control groups, at 22.3% and 16.31%, respectively (p=0.000). Specifically, the heterozygous 8/9 genotype was significantly less than expected in both case and control groups (Hardy-Weinberg disequilibrium, p=0.000), and significantly enriched in the NCD group (Yates corrected p=0.001). Skewed heterozygous genotypes were also detected for other allele combinations, such as 6/8 vs 6/9 across groups (p=0.000). Bioinformatics studies revealed that the number of (GCC)-repeats may change the RNA secondary structure and interaction sites across human exon 1. This STR was specifically expanded beyond 2-repeats in primates. In conclusion, we report a novel biological phenomenon in which there is indication of purifying selection against heterozygous genotypes at a STR locus in human, and skewed genotype compartment in late-onset NCD vs. controls. In view of the location of this STR in the 5′ UTR, RNA/RNA or RNA/DNA heterodimer formation of the involved genotypes and possible deleterious downstream events should be considered.

Transcription shapes 3D chromatin organization by interacting with loop-extruding cohesin complexes

Cohesin organizes mammalian interphase chromosomes by reeling chromatin fibers into dynamic loops (Banigan and Mirny, 2020; Davidson et al., 2019; Kim et al., 2019; Yatskevich et al., 2019). "Loop extrusion" is obstructed when cohesin encounters a properly oriented CTCF protein (Busslinger et al., 2017; de Wit et al., 2015; Fudenberg et al., 2016; Nora et al., 2017; Sanborn et al., 2015; Wutz et al., 2017), and recent work indicates that other factors, such as the replicative helicase MCM (Dequeker et al., 2020), can also act as barriers to loop extrusion. It has been proposed that transcription relocalizes (Busslinger et al., 2017; Glynn et al., 2004; Lengronne et al., 2004) or interferes with cohesin (Heinz et al., 2018; Jeppsson et al., 2020; Valton et al., 2021; S. Zhang et al., 2021), and that active transcription start sites function as cohesin loading sites (Busslinger et al., 2017; Kagey et al., 2010; Zhu et al., 2021; Zuin et al., 2014), but how these effects, and transcription in general, shape chromatin is unknown. To determine whether transcription can modulate loop extrusion, we studied cells in which the primary extrusion barriers could be removed by CTCF depletion and cohesin's residence time and abundance on chromatin could be increased by Wapl knockout. We found evidence that transcription directly interacts with loop extrusion through a novel "moving barrier" mechanism, but not by loading cohesin at active promoters. Hi-C experiments showed intricate, cohesin-dependent genomic contact patterns near actively transcribed genes, and in CTCF-Wapl double knockout (DKO) cells (Busslinger et al., 2017), genomic contacts were enriched between sites of transcription-driven cohesin localization ("cohesin islands"). Similar patterns also emerged in polymer simulations in which transcribing RNA polymerases (RNAPs) acted as "moving barriers" by impeding, slowing, or pushing loop-extruding cohesins. The model predicts that cohesin does not load preferentially at promoters and instead accumulates at TSSs due to the barrier function of RNAPs. We tested this prediction by new ChIP-seq experiments, which revealed that the "cohesin loader" Nipbl (Ciosk et al., 2000) co-localizes with cohesin, but, unlike in previous reports (Busslinger et al., 2017; Kagey et al., 2010; Zhu et al., 2021; Zuin et al., 2014), Nipbl did not accumulate at active promoters. We propose that RNAP acts as a new type of barrier to loop extrusion that, unlike CTCF, is not stationary in its precise genomic position, but is itself dynamically translocating and relocalizes cohesin along DNA. In this way, loop extrusion could enable translocating RNAPs to maintain contacts with distal regulatory elements, allowing transcriptional activity to shape genomic functional organization.

Cap analysis of gene expression reveals alternative promoter usage in a rat model of hypertension

The role of alternative promoter usage in tissue-specific gene expression has been well established; however, its role in complex diseases is poorly understood. We performed cap analysis of gene expression (CAGE) sequencing from the left ventricle of a rat model of hypertension, the spontaneously hypertensive rat (SHR), and a normotensive strain, Brown Norway to understand the role of alternative promoter usage in complex disease. We identified 26,560 CAGE-defined transcription start sites in the rat left ventricle, including 1,970 novel cardiac transcription start sites. We identified 28 genes with alternative promoter usage between SHR and Brown Norway, which could lead to protein isoforms differing at the amino terminus between two strains and 475 promoter switching events altering the length of the 5′ UTR. We found that the shift in Insr promoter usage was significantly associated with insulin levels and blood pressure within a panel of HXB/BXH recombinant inbred rat strains, suggesting that hyperinsulinemia due to insulin resistance might lead to hypertension in SHR. Our study provides a preliminary evidence of alternative promoter usage in complex diseases.

Multi-omics reveals principles of gene regulation and pervasive non-productive transcription in the human cytomegalovirus genome

For decades, human cytomegalovirus (HCMV) was thought to express ≈200 viral proteins during lytic infection. In recent years, systems biology approaches uncovered hundreds of additional viral gene products and suggested thousands of viral sites of transcription initiation. Despite all available data, the molecular mechanisms of HCMV gene regulation remain poorly understood. Here, we provide a unifying model of productive HCMV gene expression employing transcription start site profiling combined with metabolic RNA labeling as well as integrative computational analysis of previously published big data. This approach defined the expression of >2,600 high confidence viral transcripts and explained the complex kinetics of viral protein expression by cumulative effects of translation of incoming virion-associated RNA, multiple transcription start sites with distinct kinetics per viral open reading frame, and differences in viral protein stability. Most importantly, we identify pervasive transcription of transient RNAs as a common feature of this large DNA virus with its human host.

STREAMING-tag system reveals spatiotemporal relationships between transcriptional regulatory factors and transcriptional activity

Transcription is a dynamic process that stochastically switches between the ON and OFF states. To detect the dynamic relationship among protein clusters of RNA polymerase II (RNAPII) and coactivators, gene loci, and transcriptional activity, we inserted an MS2 repeat, a TetO repeat, and inteins with a selection marker just downstream of the transcription start site (TSS). By optimizing the individual elements, we have developed the Spliced TetO REpeAt, MS2 repeat, and INtein sandwiched reporter Gene tag (STREAMING-tag) system. Clusters of RNAPII and BRD4 were observed proximally to the TSS of Nanog when the gene was transcribed in mouse embryonic stem cells. In contrast, clusters of MED19 and MED22 Mediator subunits were constitutively located near the TSS. Thus, the STREAMING-tag system revealed the spatiotemporal relationships between transcriptional activity and protein clusters near the gene. This powerful tool is useful for quantitatively understanding dynamic transcriptional regulation in living cells.

Integration of high-resolution promoter profiling assays reveals novel, cell type-specific transcription start sites across 115 human cell and tissue types

Accurate transcription start site (TSS) annotations are essential for understanding transcriptional regulation and its role in human disease. Gene collections such as GENCODE contain annotations for tens of thousands of TSSs, but not all of these annotations are experimentally validated, nor do they contain information on cell type-specific usage. Therefore, we sought to generate a collection of experimentally validated TSSs by integrating RNA Annotation and Mapping of Promoters for the Analysis of Gene Expression (RAMPAGE) data from 115 cell and tissue types, which resulted in a collection of approximately 50 thousand representative RAMPAGE peaks. These peaks were primarily proximal to GENCODE-annotated TSSs and were concordant with other transcription assays. Because RAMPAGE uses paired-end reads, we were then able to connect peaks to transcripts by analyzing the genomic positions of the 3' ends of read mates. Using this paired-end information, we classified the vast majority (37 thousand) of our RAMPAGE peaks as verified TSSs, updating TSS annotations for 20% of GENCODE genes. We also found that these updated TSS annotations were supported by epigenomic and other transcriptomic datasets. To demonstrate the utility of this RAMPAGE rPeak collection, we intersected it with the NHGRI/EBI genome-wide association studies (GWAS) catalog and identified new candidate GWAS genes. Overall, our work demonstrates the importance of integrating experimental data to further refine TSS annotations and provides a valuable resource for the biological community.

Transcriptional and post-transcriptional regulation of young genes in plants

New genes continuously emerge from non-coding DNA or by diverging from existing genes, but most of them are rapidly lost and only a few become fixed within the population. We hypothesized that young genes are subject to transcriptional and post-transcriptional regulation to limit their expression and minimize their exposure to purifying selection. We found that young genes in rice have relatively low expression levels, which can be attributed to distal enhancers, and closed chromatin conformation at their transcription start sites (TSS). The chromatin in TSS regions can be re-modeled in response to abiotic stress, indicating conditional expression of young genes. Furthermore, transcripts of young genes in Arabidopsis tend to be targeted by nonsense-mediated RNA decay, presenting another layer of regulation limiting their expression. Together, these data suggest that transcriptional and post-transcriptional mechanisms contribute to the conditional expression of young genes, which may alleviate purging selection while providing an opportunity for phenotypic exposure and functionalization.

Fusaricidin Biosynthesis Is Controlled via a KinB-Spo0A-AbrB Signal Pathway in Paenibacillus polymyxa WLY78

Fusaricidins produced by Paenibacillus polymyxa are important lipopeptide antibiotics against fungi. The fusGFEDCBA (fusaricidin biosynthesis) operon is responsible for synthesis of fusaricidins. However, the regulation mechanisms of fusaricidin biosynthesis remain to be fully clarified. In this study, we revealed that fusaricidin production is controlled by a complex regulatory network including KinB-Spo0A-AbrB. Evidence suggested that the regulator AbrB represses the transcription of the fus gene cluster by direct binding to the fus promoter, in which the sequences (5′-AATTTTAAAATAAATTTTGTGATTT-3′) located from −136 to −112 bp relative to the transcription start site is required for this repression. Spo0A binds to the abrB promoter that contains the Spo0A-binding sequences (5′-TGTCGAA-3′, 0A box) and in turn prevents the further transcription of abrB. The decreasing concentration of AbrB allows for the derepression of the fus promoter repressed by AbrB. The genome of P. polymyxa WLY78 contains two orthologs (named Kin1508 and Kin4833) of Bacillus subtilis KinB, but only Kin4833 activates sporulation and fusaricidin production, indicating that this kinase may be involved in phosphorylating Spo0A to initiate sporulation and regulate the abrB transcription. Our results reveal that Kin4833 (KinB), Spo0A, and AbrB are involved in regulation of fusaricidin production and a signaling mechanism that links fusaricidin production and sporulation. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Genome-wide identification of enhancers and transcription factors regulating the myogenic differentiation of bovine satellite cells

Background Satellite cells are the myogenic precursor cells in adult skeletal muscle. The objective of this study was to identify enhancers and transcription factors that regulate gene expression during the differentiation of bovine satellite cells into myotubes. Results Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) was performed to identify genomic regions where lysine 27 of H3 histone is acetylated (H3K27ac), i.e., active enhancers, from bovine satellite cells before and during differentiation into myotubes. A total of 19,027 and 47,669 H3K27ac-marked enhancers were consistently identified from two biological replicates of before- and during-differentiation bovine satellite cells, respectively. Of these enhancers, 5882 were specific to before-differentiation, 35,723 to during-differentiation, and 13,199 common to before- and during-differentiation bovine satellite cells. Whereas most of the before- or during-differentiation-specific H3K27ac-marked enhancers were located distally to the transcription start site, the enhancers common to before- and during-differentiation were located both distally and proximally to the transcription start site. The three sets of H3K27ac-marked enhancers were associated with functionally different genes and enriched with different transcription factor binding sites. Specifically, many of the H3K27ac-marked enhancers specific to during-differentiation bovine satellite cells were associated with genes involved in muscle structure and development, and were enriched with binding sites for the MyoD, AP-1, KLF, TEAD, and MEF2 families of transcription factors. A positive role was validated for Fos and FosB, two AP-1 family transcription factors, in the differentiation of bovine satellite cells into myotubes by siRNA-mediated knockdown. Conclusions Tens of thousands of H3K27ac-marked active enhancers have been identified from bovine satellite cells before or during differentiation. These enhancers contain binding sites not only for transcription factors whose role in satellite cell differentiation is well known but also for transcription factors whose role in satellite cell differentiation is unknown. These enhancers and transcription factors are valuable resources for understanding the complex mechanism that mediates gene expression during satellite cell differentiation. Because satellite cell differentiation is a key step in skeletal muscle growth, the enhancers, the transcription factors, and their target genes identified in this study are also valuable resources for identifying and interpreting skeletal muscle trait-associated DNA variants in cattle.

Structural basis of INTAC-regulated transcription

For the majority of expressed eukaryotic genes, RNA polymerase II (Pol II) forms a paused elongation complex (PEC) and undergoes promoter-proximal pausing downstream of the transcription start site. The polymerase either proceeds into productive elongation or undergoes promoter-proximal premature transcription termination. It remains incompletely understood how transcription is regulated at this stage. Here, we determined the structure of PEC bound to INTAC, an Integrator-containing PP2A complex, at near-atomic resolution. The structure shows that INTAC partially wraps around PEC through multiple contacts, permitting the memetic nascent RNA to run into substrate-entry tunnel of the endonuclease subunit INTS11 of INTAC for cleavage. Pol II C-terminal domain (CTD) winds over INTAC backbone module through multiple anchors and is suspended above the phosphatase of INTAC for dephosphorylation. Biochemical analysis shows that INTAC-PEC association requires unphosphorylated CTD and could tolerate CTD phosphorylation, suggesting an INTAC-mediated persistent CTD dephosphorylation followed by reinforcement of the INTAC-PEC complex. Our study reveals how INTAC binds PEC and orchestrates RNA cleavage and CTD dephosphorylation, two critical events in generating premature transcription termination.