Wenjing Yang

PfSETvs methylation of histone H3K36 represses virulence genes in Plasmodium falciparum

The variant antigen Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), which is expressed on the surface of P. falciparum-infected red blood cells, is a critical virulence factor for malaria. Each parasite has 60 antigenically distinct var genes that each code for a different PfEMP1 protein. During infection the clonal parasite population expresses only one gene at a time before switching to the expression of a new variant antigen as an immune-evasion mechanism to avoid the host antibody response. The mechanism by which 59 of the 60 var genes are silenced remains largely unknown. Here we show that knocking out the P. falciparum variant-silencing SET gene (here termed PfSETvs), which encodes an orthologue of Drosophila melanogaster ASH1 and controls histone H3 lysine 36 trimethylation (H3K36me3) on var genes, results in the transcription of virtually all var genes in the single parasite nuclei and their expression as proteins on the surface of individual infected red blood cells. PfSETvs-dependent H3K36me3 is present along the entire gene body, including the transcription start site, to silence var genes. With low occupancy of PfSETvs at both the transcription start site of var genes and the intronic promoter, expression of var genes coincides with transcription of their corresponding antisense long noncoding RNA. These results uncover a previously unknown role of PfSETvs-dependent H3K36me3 in silencing var genes in P. falciparum that might provide a general mechanism by which orthologues of PfSETvs repress gene expression in other eukaryotes. PfSETvs knockout parasites expressing all PfEMP1 proteins may also be applied to the development of a malaria vaccine.

RNA polymerase II–associated factor 1 regulates the release and phosphorylation of paused RNA polymerase II

“Please release me, let me go.” RNA polymerase II (Pol II) is the principal protein complex required for gene transcription in metazoan cells. Many genes have a “paused” Pol II near their promoters, waiting to be released so they can start messenger RNA synthesis. Yu et al. show that Pol II–associated factor 1 (PAF1) plays a central role in regulating the activation of these paused Pol II complexes. The positive transcription elongation factor b helps recruit PAF1 to the paused Pol II. This facilitates the phosphorylation of Pol II on its C-terminal domain, freeing it to start transcription in earnest. Science, this issue p. 1383 RNA polymerase is paused near the promoters of many genes, and Pol II–associated factor 1 plays a critical role in its release. Release of promoter-proximal paused RNA polymerase II (Pol II) during early elongation is a critical step in transcriptional regulation in metazoan cells. Paused Pol II release is thought to require the kinase activity of cyclin-dependent kinase 9 (CDK9) for the phosphorylation of DRB sensitivity–inducing factor, negative elongation factor, and C-terminal domain (CTD) serine-2 of Pol II. We found that Pol II–associated factor 1 (PAF1) is a critical regulator of paused Pol II release, that positive transcription elongation factor b (P-TEFb) directly regulates the initial recruitment of PAF1 complex (PAF1C) to genes, and that the subsequent recruitment of CDK12 is dependent on PAF1C. These findings reveal cooperativity among P-TEFb, PAF1C, and CDK12 in pausing release and Pol II CTD phosphorylation.

Histone H2B ubiquitin ligase RNF20 is required for MLL-rearranged leukemia

Mixed-lineage leukemia (MLL) fusions are potent oncogenes that initiate aggressive forms of acute leukemia. As aberrant transcriptional regulators, MLL-fusion proteins alter gene expression in hematopoietic cells through interactions with the histone H3 lysine 79 (H3K79) methyltransferase DOT1L. Notably, interference with MLL-fusion cofactors like DOT1L is an emerging therapeutic strategy in this disease. Here, we identify the histone H2B E3 ubiquitin ligase ring finger protein 20 (RNF20) as an additional chromatin regulator that is necessary for MLL-fusion–mediated leukemogenesis. Suppressing the expression of Rnf20 in diverse models of MLL-rearranged leukemia leads to inhibition of cell proliferation, under tissue culture conditions as well as in vivo. Rnf20 knockdown leads to reduced expression of MLL-fusion target genes, effects resembling Dot1l inhibition. Using ChIP-seq, we found that H2B ubiquitination is enriched in the body of MLL-fusion target genes, correlating with sites of H3K79 methylation and transcription elongation. Furthermore, Rnf20 is required to maintain local levels of H3K79 methylation by Dot1l at Hoxa9 and Meis1. These findings support a model whereby cotranscriptional recruitment of Rnf20 at MLL-fusion target genes leads to amplification of Dot1l-mediated H3K79 methylation, thereby rendering leukemia cells dependent on Rnf20 to maintain their oncogenic transcriptional program.

Identification of an ABCB1 (P-glycoprotein)-positive carfilzomib-resistant myeloma subpopulation by the pluripotent stem cell fluorescent dye CDy1

Multiple myeloma (MM) is characterized by the malignant expansion of differentiated plasma cells. Although many chemotherapeutic agents display cytotoxic activity toward MM cells, patients inevitably succumb to their disease because the tumor cells become resistant to the anticancer drugs. The cancer stem cell hypothesis postulates that a small subpopulation of chemotherapy‐resistant cancer cells is responsible for propagation of the tumor. Herein we report that efflux of the pluripotent stem cell dye CDy1 identifies a subpopulation in MM cell lines characterized by increased expression of P‐glycoprotein, a member of the ABC (ATP‐binding cassette) superfamily of transporters encoded by ABCB1. We also demonstrate that ABCB1‐overexpressing MM cells are resistant to the second‐generation proteasome inhibitor carfilzomib that recently received accelerated approval for the treatment of therapy‐refractive MM by the U.S. Food and Drug Administration. Moreover, increased resistance to carfilzomib in sensitive MM cells following drug selection was associated with upregulation of ABCB1 cell‐surface expression which correlated with increased transporter activity as measured by CDy1 efflux. We further show that chemosensitization of MM cells to carfilzomib could be achieved in vitro by cotreatment with vismodegib, a hedgehog pathway antagonist which is currently in MM clinical trials. CDy1 efflux may therefore be a useful assay to determine whether high expression of ABCB1 is predictive of poor clinical responses in MM patients treated with carfilzomib. Our data also suggest that inclusion of vismodegib might be a potential strategy to reverse ABCB1‐mediated drug resistance should it occur. Am. J. Hematol. 88:265–272, 2013.

Distinct polyadenylation landscapes of diverse human tissues revealed by a modified PA-seq strategy

BackgroundPolyadenylation is a key regulatory step in eukaryotic gene expression and one of the major contributors of transcriptome diversity. Aberrant polyadenylation often associates with expression defects and leads to human diseases.ResultsTo better understand global polyadenylation regulation, we have developed a polyadenylation sequencing (PA-seq) approach. By profiling polyadenylation events in 13 human tissues, we found that alternative cleavage and polyadenylation (APA) is prevalent in both protein-coding and noncoding genes. In addition, APA usage, similar to gene expression profiling, exhibits tissue-specific signatures and is sufficient for determining tissue origin. A 3′ untranslated region shortening index (USI) was further developed for genes with tandem APA sites. Strikingly, the results showed that different tissues exhibit distinct patterns of shortening and/or lengthening of 3′ untranslated regions, suggesting the intimate involvement of APA in establishing tissue or cell identity.ConclusionsThis study provides a comprehensive resource to uncover regulated polyadenylation events in human tissues and to characterize the underlying regulatory mechanism.

Global intron retention mediated gene regulation during CD4+ T cell activation

T cell activation is a well-established model for studying cellular responses to exogenous stimulation. Using strand-specific RNA-seq, we observed that intron retention is prevalent in polyadenylated transcripts in resting CD4+ T cells and is significantly reduced upon T cell activation. Several lines of evidence suggest that intron-retained transcripts are less stable than fully spliced transcripts. Strikingly, the decrease in intron retention (IR) levels correlate with the increase in steady-state mRNA levels. Further, the majority of the genes upregulated in activated T cells are accompanied by a significant reduction in IR. Of these 1583 genes, 185 genes are predominantly regulated at the IR level, and highly enriched in the proteasome pathway, which is essential for proper T cell proliferation and cytokine release. These observations were corroborated in both human and mouse CD4+ T cells. Our study revealed a novel post-transcriptional regulatory mechanism that may potentially contribute to coordinated and/or quick cellular responses to extracellular stimuli such as an acute infection.

A Viral Genome Landscape of RNA Polyadenylation from KSHV Latent to Lytic Infection

RNA polyadenylation (pA) is one of the major steps in regulation of gene expression at the posttranscriptional level. In this report, a genome landscape of pA sites of viral transcripts in B lymphocytes with Kaposi sarcoma-associated herpesvirus (KSHV) infection was constructed using a modified PA-seq strategy. We identified 67 unique pA sites, of which 55 could be assigned for expression of annotated ∼90 KSHV genes. Among the assigned pA sites, twenty are for expression of individual single genes and the rest for multiple genes (average 2.7 genes per pA site) in cluster-gene loci of the genome. A few novel viral pA sites that could not be assigned to any known KSHV genes are often positioned in the antisense strand to ORF8, ORF21, ORF34, K8 and ORF50, and their associated antisense mRNAs to ORF21, ORF34 and K8 could be verified by 3′RACE. The usage of each mapped pA site correlates to its peak size, the larger (broad and wide) peak size, the more usage and thus, the higher expression of the pA site-associated gene(s). Similar to mammalian transcripts, KSHV RNA polyadenylation employs two major poly(A) signals, AAUAAA and AUUAAA, and is regulated by conservation of cis-elements flanking the mapped pA sites. Moreover, we found two or more alternative pA sites downstream of ORF54, K2 (vIL6), K9 (vIRF1), K10.5 (vIRF3), K11 (vIRF2), K12 (Kaposin A), T1.5, and PAN genes and experimentally validated the alternative polyadenylation for the expression of KSHV ORF54, K11, and T1.5 transcripts. Together, our data provide not only a comprehensive pA site landscape for understanding KSHV genome structure and gene expression, but also the first evidence of alternative polyadenylation as another layer of posttranscriptional regulation in viral gene expression.

An Adult Mouse Thyroid Side Population Cell Line that Exhibits Enriched Epithelial–Mesenchymal Transition

BACKGROUND Studies of thyroid stem/progenitor cells have been hampered due to the small organ size and lack of tissue, which limits the yield of these cells. A continuous source that allows the study and characterization of thyroid stem/progenitor cells is desired to push the field forward. METHOD A cell line was established from Hoechst-resistant side population cells derived from mouse thyroid that were previously shown to contain stem/progenitor-like cells. Characterization of these cells were carried out by using in vitro two- and three-dimensional cultures and in vivo reconstitution of mice after orthotopic or intravenous injection, in conjunction with quantitative reverse transcription polymerase chain reaction, Western blotting, immunohisto(cyto)chemistry/immunofluorescence, and RNA seq analysis. RESULTS These cells were named SPTL (side population cell-derived thyroid cell line). Under low serum culturing conditions, SPTL cells expressed the thyroid differentiation marker NKX2-1, a transcription factor critical for thyroid differentiation and function, while no expression of other thyroid differentiation marker genes were observed. SPTL cells formed follicle-like structures in Matrigel® cultures, which did not express thyroid differentiation marker genes. In mouse models of orthotopic and intravenous injection, the latter following partial thyroidectomy, a few SPTL cells were found in part of the follicles, most of which expressed NKX2-1. SPTL cells highly express genes involved in epithelial-mesenchymal transition, as demonstrated by RNA seq analysis, and exhibit a gene-expression pattern similar to anaplastic thyroid carcinoma. CONCLUSION These results demonstrate that SPTL cells have the capacity to differentiate into thyroid to a limited degree. SPTL cells may provide an excellent tool to study stem cells, including cancer stem cells of the thyroid.

NRF1 coordinates with DNA methylation to regulate spermatogenesis

Spermatogenesis is a highly coordinated process that requires tightly regulated gene expression programmed by transcription factors and epigenetic modifiers. In this study, we found that nuclear respiratory factor (NRF)‐1, a key transcription factor for mitochondrial biogenesis, cooperated with DNA methylation to directly regulate the expression of multiple germ cell–specific genes, including Asz1. In addition, conditional ablation of NRF1 in gonocytes dramatically down‐regulated these germline genes, blocked germ cell proliferation, and subsequently led to male infertility in mice. Our data highlight a precise crosstalk between transcriptional regulation by NRF1 and epigenetic modulation during germ cell development and unequivocally demonstrate a novel role of NRF1 in spermatogenesis.—Wang, J., Tang, C., Wang, Q., Su, J., Ni, T., Yang, W., Wang, Y., Chen, W., Liu, X., Wang, S., Zhang, J., Song, H., Zhu, J., Wang, Y. NRF1 coordinates with DNA methylation to regulate spermatogenesis. FASEB J. 31, 4959–4970 (2017). www.fasebj.org

p53 as a Therapeutic Target in T-ALL

TP53 is a central hub integrating stress signals from oncogenic genetic lesions and cytotoxic anti-cancer agents. The function of p53 as a regulator of transcription is well documented. More recently it was shown to directly interact with BCL2 family members and induce mitochondrial cell death. Stress-induced activation of p53 leads to cell cycle arrest that allows metabolic adjustments and repair mechanisms to take place prior to the next cycle; p53 may also induce senescence or apoptosis depending on the strength and nature of stress stimuli and/or cell type. In 50% of human cancers, the TP53 gene is deleted or mutated with a high proportion of gain of oncogenic function mutations. It is noteworthy therefore that TP53 is rarely mutated in T-ALL. However, its tumor suppressor activity is circumvented by genetic lesions. We will discuss here the most frequent T-ALL genetic abnormalities of INK4A/ARF, NOTCH1 and PTEN genes and how they affect TP53 expression and function. Current understanding of the signaling pathways governed by these oncogenes is advanced enough to find points of intersection with p53 downstream targets and attempt to translate the accumulated knowledge to the clinic. In addition, we will discuss the results of our analysis of publicly available expression profiling data indicating the existence of a TP53-anchored transcriptional program targeted by T-ALL oncogenes such as NOTCH, MYC and TLX1 in primary leukemic cells and how it can be exploited for cancer intervention. Overall, the goal of this chapter is to describe how T-ALL pathobiology affects the p53interacting network, highlighting some new potential therapeutic targets as well as some still unresolved questions.