Recurrent integration of human papillomavirus genomes at transcriptional regulatory hubs

Oncogenic human papillomavirus (HPV) genomes are often integrated into host chromosomes in HPV-associated cancers. HPV genomes are integrated either as a single copy or as tandem repeats of viral DNA interspersed with, or without, host DNA. Integration occurs frequently in common fragile sites susceptible to tandem repeat formation and the flanking or interspersed host DNA often contains transcriptional enhancer elements. When co-amplified with the viral genome, these enhancers can form super-enhancer-like elements that drive high viral oncogene expression. Here we compiled highly curated datasets of HPV integration sites in cervical (CESC) and head and neck squamous cell carcinoma (HNSCC) cancers, and assessed the number of breakpoints, viral transcriptional activity, and host genome copy number at each insertion site. Tumors frequently contained multiple distinct HPV integration sites but often only one “driver” site that expressed viral RNA. As common fragile sites and active enhancer elements are cell-type-specific, we mapped these regions in cervical cell lines using FANCD2 and Brd4/H3K27ac ChIP-seq, respectively. Large enhancer clusters, or super-enhancers, were also defined using the Brd4/H3K27ac ChIP-seq dataset. HPV integration breakpoints were enriched at both FANCD2-associated fragile sites and enhancer-rich regions, and frequently showed adjacent focal DNA amplification in CESC samples. We identified recurrent integration “hotspots” that were enriched for super-enhancers, some of which function as regulatory hubs for cell-identity genes. We propose that during persistent infection, extrachromosomal HPV minichromosomes associate with these transcriptional epicenters and accidental integration could promote viral oncogene expression and carcinogenesis.

HIC2 Controls Developmental Hemoglobin Switching By Repressing BCL11A Transcription

One of the oldest and most deeply studied problems in developmental gene expression is the switch from fetal to adult type hemoglobin production in red blood cell precursors. Interest in this question has been fueled by its relevance to genetic blood disorders such as sickle cell disease (SCD) and thalassemia. BCL11A is a transcriptional repressor that is thought to directly silence the fetal β-type globin (HBG1/2) genes in adult erythroid cells. Transcriptome and RNA polymerase II profiling indicate that the BCL11A gene is transcribed considerably more highly in adult erythroblasts compared to fetal cells, accounting in large part for corresponding changes in BCL11A protein levels. Yet, the mechanism governing BCL11A developmental regulation is still unclear. To identify novel regulators of the fetal-to-adult globin switch, we interrogated our recent CRISPR based genetic screens that employed single guide RNAs (sgRNAs) targeting transcription factors (Huang et al., Blood, 2020) and uncovered HIC2, a penta-dactyl zinc finger DNA binding protein bearing a BTB/POZ domain as a novel regulator of hemoglobin switching. HIC2 is expressed more highly in fetal erythroblasts compared to adult cells, a pattern inverse to that of BCL11A. Overexpression (OE) of HIC2 in the adult type erythroid HUDEP2 cell line stimulated the expression of 322 genes while impairing that of 224 genes (FDR < 0.01 and fold change ≥ 2). The most highly upregulated genes (>150-fold) were HBG1/2. Upregulation was accompanied by gains in chromatin accessibility and histone H3K27acetylation of HBG1/2, and increased chromatin contacts between the distal globin gene enhancer (LCR) and the HBG1/2 genes. Overexpression of HIC2 in primary human erythroblasts also significantly increased HBG1/2 mRNA and protein levels, sufficient to reduce cell sickling in SCD patient-derived erythroid cells. HIC2 OE lowered BCL11A mature and pre-mRNA production, indicating that HIC2 attenuates BCL11A transcription. Forced expression of BCL11A restored HBG1/2 silencing in HIC2 OE HUDEP2 cells, suggesting that BCL11A repression accounts for the effects of HIC2 on fetal globin genes. ChIP-seq revealed a strong HIC2 binding peak at the erythroid BCL11A +55 enhancer. HIC2 OE reduced chromatin accessibility and H3K27acetylation of the +55 enhancer, as well as the enhancer-promoter contacts, suggesting that HIC2 directly decommissions the enhancer to attenuate BCL11A transcription. The BCL11A +55 enhancer contains two consensus HIC2 binding motifs under the HIC2 peak adjacent to GATA:E-box and GATA motifs. CRISPR-mediated mutagenesis of both HIC2 motifs raised BCL11A basal level transcription and diminished the ability of overexpressed HIC2 to repress BCL11A transcription. Notably, HIC2 OE impaired binding of transcription factor GATA1 at the +55 enhancer, suggesting that this enhancer is under developmental control. Indeed, GATA1 binding and chromatin accessibility of +55 enhancer were virtually undetectable in HUDEP1 cells, which represent a more fetal-like state. CRISPR-mediated depletion of HIC2 in HUDEP1 cells reversed this pattern with gains in GATA1 binding, chromatin accessibility, and BCL11A transcription. In sum, HIC2 emerges as a critical regulator of hemoglobin switching that operates by imposing developmental stage-specific control onto a BCL11A transcriptional enhancer. Disclosures Blobel: Fulcrum therapeutics: Consultancy; Pfizer: Research Funding.

Recurrent Integration of Human Papillomavirus Genomes at Transcriptional Regulatory Hubs

Oncogenic human papillomavirus (HPV) genomes are often integrated into host chromosomes in HPV-associated cancers. HPV genomes are integrated either as a single copy, or as tandem repeats of viral DNA interspersed with, or without, host DNA. Integration occurs frequently in common fragile sites susceptible to tandem repeat formation, and the flanking or interspersed host DNA often contains transcriptional enhancer elements. When co-amplified with the viral genome, these enhancers can form super-enhancer-like elements that drive high viral oncogene expression. Here, we compiled highly curated datasets of HPV integration sites in cervical (CESC) and head and neck squamous cell carcinoma (HNSCC) cancers and assessed the number of breakpoints, viral transcriptional activity, and host genome copy number at each insertion site. Tumors frequently contained multiple distinct HPV integration sites, but often only one driver site that expressed viral RNA. Since common fragile sites and active enhancer elements are cell-type specific, we mapped these regions in cervical cell lines using FANCD2 and Brd4/H3K27ac ChIP-seq, respectively. Large enhancer clusters, or super-enhancers, were also defined using the Brd4/H3K27ac ChIP-seq dataset. HPV integration breakpoints were enriched at both FANCD2-associated fragile sites, and enhancer-rich regions, and frequently showed adjacent focal DNA amplification in CESC samples. We identified recurrent integration hotspots that were enriched for super-enhancers, some of which function as regulatory hubs for cell-identity genes. We propose that during persistent infection, extrachromosomal HPV minichromosomes associate with these transcriptional epicenters, and accidental integration could promote viral oncogene expression and carcinogenesis.

Six1 Promotes Skeletal Muscle Thyroid Hormone Response through Regulation of the MCT10 Transporter

The Six1 transcription factor is implicated in controlling the development of several tissue types, notably skeletal muscle. Six1 also contributes to muscle metabolism and its activity is associated with the fast-twitch, glycolytic phenotype. Six1 regulates the expression of certain genes of the fast muscle program by directly stimulating their transcription or indirectly acting through a long non-coding RNA. Under the hypothesis that additional mechanisms of action might be at play, a combined analysis of gene expression profiling and genome-wide location analysis data was performed. The Slc16a10 gene, encoding the thyroid hormone transmembrane transporter MCT10, was identified as a gene with a transcriptional enhancer directly bound by Six1 and requiring Six1 activity for full expression in adult mouse tibialis anterior, a predominantly fast-twitch muscle. Of the various thyroid hormone transporters, MCT10 mRNA was found to be the most abundant in skeletal muscle, and to have a stronger expression in fast-twitch compared to slow-twitch muscle groups. Loss-of-function of MCT10 in the tibialis anterior recapitulated the effect of Six1 on the expression of fast-twitch muscle genes and led to lower activity of a thyroid hormone receptor-dependent reporter gene. These results shed light on the molecular mechanisms controlling the tissue expression profile of MCT10 and identify modulation of the thyroid hormone signaling pathway as an additional mechanism by which Six1 influences skeletal muscle metabolism.

Regulation of HPV18 Genome Replication, Establishment and Persistence by Sequences in the Viral Upstream Regulatory Region

During persistent human papillomavirus infection, the viral genome replicates as an extrachromosomal plasmid that is efficiently partitioned to daughter cells during cell division. We have previously shown that an element which overlaps the HPV18 transcriptional enhancer promotes stable DNA replication of replicons containing the viral replication origin. Here we perform comprehensive analyses to elucidate the function of this maintenance element. We conclude that no unique element or binding site in this region is absolutely required for persistent replication and partitioning, and instead propose that the overall chromatin architecture of this region is important to promote efficient use of the replication origin. These results have important implications on the genome partitioning mechanism of papillomaviruses. Importance Persistent infection with oncogenic HPVs is responsible for ∼5% human cancers. The viral DNA replicates as an extrachromosomal plasmid and is partitioned to daughter cells in dividing keratinocytes. Using a complementation assay that allows us to separate viral transcription and replication, we provide insight into viral sequences that are required for long term replication and persistence in keratinocytes. Understanding how viral genomes replicate persistently for such long periods of time will guide the development of anti-viral therapies.

POS1223 RAS GUANYL RELEASING PROTEIN 1 (RASGRP1) IN PERIPHERAL B CELLS LINKS RA TO COVID-19

Background:Changes in the B cell subpopulations is a hallmark of the antiviral response against SARS-CoV-2 and is associated with COVID-19 severity (1). Recently our group showed common derangement observed in rheumatoid arthritis (RA) and COVID-19 (2). In RA, synovium attracts potentially autoreactive—B cells and plasma cells that play a central role in RA pathogenesis (3). We were interested to know the similarity in B cell’s transcriptomic changes specific to RA and COVID-19.Objectives:Identify similar upregulated genes in synovium and B cells in RA and at the same time are differentially expressed in B cells infected with SARS-CoV-2 or from COVID-19 patients.Methods:RNAseq dataset (GSE89408) of (218) samples isolated from joint synovial biopsies from subjects with and without rheumatoid arthritis were retrieved from GEO online database. Differentially expressed genes (DRGs) specific to RA were identified after exclusion of those upregulated in Osteoarthritis or other joint condition samples in the same dataset. The RA specific genes were intersected with DEGs between B cells from healthy versus RA as extracted from (GSE110999) dataset. The shortlisted genes specifically upregulated in B cells of RA were identified and were explored in B cells COVID-19 transcriptome datasets using (https://metascape.org/COVID).Results:60 genes were found to be specifically upregulated in RA synovium and B cells and are changed in B cells infected with SARS-CoV-2 or from COVID-19 patients, Figure (1-A). Those genes were involved in interferon signaling, antiviral and immune cell activation. RASGRP1 was common between B cells of RA and COVID-19 and might play a role in the pathogenesis of both, Figure (1-B). RASGRP1 controls ERK/MAPK kinase cascade needed in B-/T-cell differentiation and development. It is vital to protect against viral infection and the autoimmune associated proliferation of activated T-cells like RA (4). We checked its level in another dataset (GSE152641) of the whole blood RNASeq of 62 COVID-19 patients and 24 healthy controls. RASGRP1 was significantly down in COVID-19 compared to healthy control, Figure (1-C).Conclusion:SARS-CoV-2 impair B and T’s cells’ immune response through its action on RASGRP1 and that can be a novel mechanistic explanation of how the virus decreases immune cells and impair the B cell’s humoral immunity.References:[1]Sosa-Hernández VA, Torres-Ruíz J, Cervantes-Díaz R, Romero-Ramírez S, Páez-Franco JC, Meza-Sánchez DE, et al. B Cell Subsets as Severity-Associated Signatures in COVID-19 Patients. Frontiers in Immunology. 2020;11(3244).[2]Hachim MY, Hachim IY, Naeem KB, Hannawi H, Al Salmi I, Hannawi S. C-C chemokine receptor type 5 links COVID-19, rheumatoid arthritis, and Hydroxychloroquine: in silico analysis. Translational Medicine Communications. 2020;5(1):14.[3]Doorenspleet ME, Klarenbeek PL, de Hair MJ, van Schaik BD, Esveldt RE, van Kampen AH, et al. Rheumatoid arthritis synovial tissue harbours dominant B-cell and plasma-cell clones associated with autoreactivity. Ann Rheum Dis. 2014;73(4):756-62.[4]Molineros JE, Singh B, Terao C, Okada Y, Kaplan J, McDaniel B, et al. Mechanistic Characterization of RASGRP1 Variants Identifies an hnRNP-K-Regulated Transcriptional Enhancer Contributing to SLE Susceptibility. Frontiers in Immunology. 2019;10(1066).Disclosure of Interests:None declared

A Transcription Start Site Map in Human Pancreatic Islets Reveals Functional Regulatory Signatures

Identifying the tissue-specific molecular signatures of active regulatory elements is critical to understand gene regulatory mechanisms. Here, we identify transcription start sites (TSS) using cap analysis of gene expression (CAGE) across 57 human pancreatic islet samples. We identify 9,954 reproducible CAGE tag clusters (TCs), ~20% of which are islet-specific and occur mostly distal to known gene TSSs. We integrated islet CAGE data with histone modification and chromatin accessibility profiles to identify epigenomic signatures of transcription initiation. Using a massively parallel reporter assay, we validated the transcriptional enhancer activity for 2,279 of 3,378 (~68%) tested islet CAGE elements (5% FDR). TCs within accessible enhancers show higher enrichment to overlap type 2 diabetes genome-wide association study (GWAS) signals than existing islet annotations, which emphasizes the utility of mapping CAGE profiles in disease-relevant tissue. This work provides a high-resolution map of transcriptional initiation in human pancreatic islets with utility for dissecting active enhancers at GWAS loci.

A Transcription Start Site Map in Human Pancreatic Islets Reveals Functional Regulatory Signatures

Identifying the tissue-specific molecular signatures of active regulatory elements is critical to understand gene regulatory mechanisms. Here, we identify transcription start sites (TSS) using cap analysis of gene expression (CAGE) across 57 human pancreatic islet samples. We identify 9,954 reproducible CAGE tag clusters (TCs), ~20% of which are islet-specific and occur mostly distal to known gene TSSs. We integrated islet CAGE data with histone modification and chromatin accessibility profiles to identify epigenomic signatures of transcription initiation. Using a massively parallel reporter assay, we validated the transcriptional enhancer activity for 2,279 of 3,378 (~68%) tested islet CAGE elements (5% FDR). TCs within accessible enhancers show higher enrichment to overlap type 2 diabetes genome-wide association study (GWAS) signals than existing islet annotations, which emphasizes the utility of mapping CAGE profiles in disease-relevant tissue. This work provides a high-resolution map of transcriptional initiation in human pancreatic islets with utility for dissecting active enhancers at GWAS loci.

RTEF-1 Inhibits Vascular Smooth Muscle Cell Calcification through Regulating Wnt/β-Catenin Signaling Pathway

Vascular calcification (VC) is highly prevailing in cardiovascular disease, diabetes mellitus, and chronic kidney disease and, when present, is associated with cardiovascular events and mortality. The osteogenic differentiation of vascular smooth muscle cells (VSMCs) is regarded as the foundation for mediating VC. Related transcriptional enhancer factor (RTEF-1), also named as transcriptional enhanced associate domain (TEAD) 4 or transcriptional enhancer factor-3 (TEF-3), is a nuclear transcriptional factor with a potent effect on cardiovascular diseases, apart from its oncogenic role in the canonical Hippo pathway. However, the role and mechanism of RTEF-1 in VC, particularly in calcification of VSMCs, are poorly understood. Our results showed that RTEF-1 was reduced in calcified VSMCs. RTEF-1 significantly ameliorated β-glycerophosphate (β-GP)-induced VSMCs calcification, as detected by alizarin red staining and calcium content assay. Also, RTEF-1 reduced alkaline phosphatase (ALP) activity and decreased expressions of osteoblast markers such as Osteocalcin and Runt-related transcription factor-2 (Runx2), but increased expression of contractile protein, including SM α-actin (α-SMA). Additionally, RTEF-1 inhibited β-GP-activated Wnt/β-catenin pathway which plays a critical role in calcification and osteogenic differentiation of VSMCs. Specifically, RTEF-1 reduced the levels of Wnt3a, p-β-catenin (Ser675), glycogen synthase kinase-3β (GSK-3β), and p-GSK-3β (Ser9), but increased the levels of p-β-catenin (Ser33/37). Also, RTEF-1 increased the ratio of p-β-catenin (Ser33/37) to β-catenin proteins and decreased the ratio of p-GSK-3β (Ser9) to GSK-3β protein. LiCl, a Wnt/β-catenin signaling activator, was observed to reverse the protective effect of RTEF-1 overexpression on VSMCs calcification induced by β-GP. Accordingly, Dickkopf-1 (Dkk1), a Wnt antagonist, attenuated the role of RTEF-1 deficiency in β-GP-induced VSMCs calcification. Taken together, we concluded that RTEF-1 ameliorated β-GP-induced calcification and osteoblastic differentiation of VSMCs by inhibiting Wnt/β-catenin signaling pathway.

Sequences in the Human papillomavirus Type 18 (HPV18) Upstream Regulatory Region regulate Viral Genome Replication, Establishment and Persistence

During persistent human papillomavirus infection, the viral genome replicates as an extrachromosomal plasmid that is efficiently partitioned to daughter cells during cell division. We have previously shown that an element which overlaps the HPV18 transcriptional enhancer promotes stable DNA replication of replicons containing the viral replication origin. Here we perform comprehensive analyses to elucidate the function of this maintenance element. We conclude that no unique element or binding site in this region is absolutely required for persistent replication and partitioning, and instead propose that the overall chromatin architecture of this region is important to promote efficient use of the replication origin. These results have important implications on the genome partitioning mechanism of papillomaviruses.