Faculty Opinions recommendation of Inhibiting gene expression at transcription start sites in chromosomal DNA with antigene RNAs.

Abstract Chinese hamster ovary (CHO) cells are widely used for producing biopharmaceuticals, and engineering gene expression in CHO is key to improving drug quality and affordability. However, engineering gene expression or activating silent genes requires accurate annotation of the underlying regulatory elements and transcription start sites (TSSs). Unfortunately, most TSSs in the published Chinese hamster genome sequence were computationally predicted and are frequently inaccurate. Here, we use nascent transcription start site sequencing methods to revise TSS annotations for 15 308 Chinese hamster genes and 3034 non-coding RNAs based on experimental data from CHO-K1 cells and 10 hamster tissues. We further capture tens of thousands of putative transcribed enhancer regions with this method. Our revised TSSs improves upon the RefSeq annotation by revealing core sequence features of gene regulation such as the TATA box and the Initiator and, as exemplified by targeting the glycosyltransferase gene Mgat3, facilitate activating silent genes by CRISPRa. Together, we envision our revised annotation and data will provide a rich resource for the CHO community, improve genome engineering efforts and aid comparative and evolutionary studies.

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The Drosophila HP1 family is associated with active gene expression across chromatin contexts

Genetics ◽

10.1093/genetics/iyab090 ◽

2021 ◽

Author(s):

John M Schoelz ◽

Justina X Feng ◽

Nicole C Riddle

Keyword(s):

Gene Expression ◽

Protein Interactions ◽

Transcriptional Activation ◽

Target Genes ◽

Family Members ◽

Transcription Start ◽

Heterochromatin Formation ◽

Transcription Start Sites ◽

Polymerase Pausing ◽

Hp1 Proteins

Abstract Drosophila Heterochromatin Protein 1a (HP1a) is essential for heterochromatin formation and is involved in transcriptional silencing. However, certain loci require HP1a to be transcribed. One model posits that HP1a acts as a transcriptional silencer within euchromatin while acting as an activator within heterochromatin. However, HP1a has been observed as an activator of a set of euchromatic genes. Therefore, it is not clear whether, or how, chromatin context informs the function of HP1 proteins. To understand the role of HP1 proteins in transcription, we examined the genome-wide binding profile of HP1a as well as two other Drosophila HP1 family members, HP1B and HP1C, to determine whether coordinated binding of these proteins is associated with specific transcriptional outcomes. We found that HP1 proteins share many of their endogenous binding targets. These genes are marked by active histone modifications and are expressed at higher levels than non-target genes in both heterochromatin and euchromatin. In addition, HP1 binding targets displayed increased RNA polymerase pausing compared to non-target genes. Specifically, co-localization of HP1B and HP1C was associated with the highest levels of polymerase pausing and gene expression. Analysis of HP1 null mutants suggests these proteins coordinate activity at transcription start sites to regulate transcription. Depletion of HP1B or HP1C alters expression of protein-coding genes bound by HP1 family members. Our data broaden understanding of the mechanism of transcriptional activation by HP1a and highlight the need to consider particular protein-protein interactions, rather than broader chromatin context, to predict impacts of HP1 at transcription start sites.

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A catalog of transcription start sites across 115 human tissue and cell types

10.1101/2021.05.12.443890 ◽

2021 ◽

Author(s):

Jill E Moore ◽

Xiao-Ou Zhang ◽

Shaimae I Elhajjajy ◽

Kaili Fan ◽

Fairlie Reese ◽

...

Keyword(s):

Gene Expression ◽

Disease Gene ◽

Cell Types ◽

Start Site ◽

Valuable Resource ◽

Cell Type ◽

Transcription Start ◽

Transcription Start Sites ◽

Cell Type Specific ◽

Gwas Catalog

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 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.

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Genome-Wide Identification of Transcription Start Sites in Two Alphaproteobacteria, Rhodobacter sphaeroides 2.4.1 and Novosphingobium aromaticivorans DSM 12444

Microbiology Resource Announcements ◽

10.1128/mra.00880-20 ◽

2020 ◽

Vol 9 (36) ◽

Author(s):

Kevin S. Myers ◽

Jessica M. Vera ◽

Kimberly C. Lemmer ◽

Alexandra M. Linz ◽

Robert Landick ◽

...

Keyword(s):

Gene Expression ◽

Rhodobacter Sphaeroides ◽

Transcription Start ◽

Future Studies ◽

Transcription Start Sites ◽

Important Resource ◽

Genome Wide

ABSTRACT Here, we report the genome-wide identification of transcription start sites (TSSs) from two Alphaproteobacteria grown under conditions that result in significant changes in gene expression. TSSs that were identified as present in one condition or both will be an important resource for future studies of these, and possibly other, Alphaproteobacteria.

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Chromatin acetylation at transcription start sites and vitamin D receptor binding regions relates to effects of 1α,25-dihydroxyvitamin D3 and histone deacetylase inhibitors on gene expression

Nucleic Acids Research ◽

10.1093/nar/gks959 ◽

2012 ◽

Vol 41 (1) ◽

pp. 110-124 ◽

Cited By ~ 50

Author(s):

Sabine Seuter ◽

Sami Heikkinen ◽

Carsten Carlberg

Keyword(s):

Gene Expression ◽

Vitamin D ◽

Histone Deacetylase ◽

Vitamin D Receptor ◽

Receptor Binding ◽

Histone Deacetylase Inhibitors ◽

Transcription Start ◽

Dihydroxyvitamin D3 ◽

Transcription Start Sites ◽

Chromatin Acetylation

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Histone modifications underlie monocyte dysregulation in patients with systemic sclerosis, underlining the treatment potential of epigenetic targeting

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2018-214295 ◽

2019 ◽

Vol 78 (4) ◽

pp. 529-538 ◽

Cited By ~ 10

Author(s):

Maarten van der Kroef ◽

Monica Castellucci ◽

Michal Mokry ◽

Marta Cossu ◽

Marianna Garonzi ◽

...

Keyword(s):

Gene Expression ◽

Systemic Sclerosis ◽

Chromatin Immunoprecipitation ◽

Transcription Start ◽

Histone Marks ◽

Transcription Start Sites ◽

Altered Gene Expression ◽

Bromodomain Proteins ◽

Altered Gene

Background and objectiveSystemic sclerosis (SSc) is a severe autoimmune disease, in which the pathogenesis is dependent on both genetic and epigenetic factors. Altered gene expression in SSc monocytes, particularly of interferon (IFN)-responsive genes, suggests their involvement in SSc development. We investigated the correlation between epigenetic histone marks and gene expression in SSc monocytes.MethodsChromatin immunoprecipitation followed by sequencing (ChIPseq) for histone marks H3K4me3 and H3K27ac was performed on monocytes of nine healthy controls and 14 patients with SSc. RNA sequencing was performed in parallel to identify aberrantly expressed genes and their correlation with the levels of H3K4me3 and H3K27ac located nearby their transcription start sites. ChIP-qPCR assays were used to verify the role of bromodomain proteins, H3K27ac and STATs on IFN-responsive gene expression.Results1046 and 534 genomic loci showed aberrant H3K4me3 and H3K27ac marks, respectively, in SSc monocytes. The expression of 381 genes was directly and significantly proportional to the levels of such chromatin marks present near their transcription start site. Genes correlated to altered histone marks were enriched for immune, IFN and antiviral pathways and presented with recurrent binding sites for IRF and STAT transcription factors at their promoters. IFNα induced the binding of STAT1 and STAT2 at the promoter of two of these genes, while blocking acetylation readers using the bromodomain BET family inhibitor JQ1 suppressed their expression.ConclusionSSc monocytes have altered chromatin marks correlating with their IFN signature. Enzymes modulating these reversible marks may provide interesting therapeutic targets to restore monocyte homeostasis to treat or even prevent SSc.

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A Chinese hamster transcription start site atlas that enables targeted editing of CHO cells

10.1101/2020.10.09.334045 ◽

2020 ◽

Author(s):

Isaac Shamie ◽

Sascha H. Duttke ◽

Karen J. la Cour Karottki ◽

Claudia Z. Han ◽

Anders H. Hansen ◽

...

Keyword(s):

Gene Expression ◽

Cho Cells ◽

Genome Engineering ◽

Chinese Hamster ◽

Regulatory Elements ◽

Tata Box ◽

Transcription Start ◽

Transcription Start Sites ◽

Silent Genes ◽

Non Coding Rnas

ABSTRACTChinese hamster ovary (CHO) cells, with their human-compatible glycosylation and high protein titers, are the most widely used cells for producing biopharmaceuticals. Engineering gene expression in CHO is key to improving drug quality and affordability. However, engineering gene expression or activating silent genes requires accurate annotation of the underlying regulatory elements and transcription start sites (TSSs). Unfortunately, most TSSs in the Chinese hamster genome were computationally predicted and are frequently inaccurate. Here, we revised TSS annotations for 15,308 Chinese hamster genes and 4,478 non-coding RNAs based on experimental data from CHO-K1 cells and 10 hamster tissues. The experimental realignment and discovery of TSSs now expose previously hidden motifs, such as the TATA box. We further demonstrate, by targeting the glycosyltransferase gene Mgat3, how accurate annotations readily facilitate activating silent genes by CRISPRa to obtain more human-like glycosylation. Together, we envision our annotation and data will provide a rich resource for the CHO community, improve genome engineering efforts and aid comparative and evolutionary studies.

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