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Investigating the role of Epstein-Barr virus lytic key regulator protein Zta in transcriptional regulation
Epstein-Barr virus (EBV) is a human herpes virus that, upon primary infection,
establishes life-long persistence in B cells (latency). One viral protein, the
immediate early lytic protein Zta (also known as BZLF1, ZEBRA, EB1, and Z)
plays a significant role in disturbing this latency and inducing a viral productive
(lytic) cycle. Expression of Zta, a basic leucine zipper transcription factor,
induces a cascade of viral lytic cycle gene expression. The activation of many
lytic genes requires the direct binding of Zta to its response elements (ZREs)
within proximal promoters of these genes. Much research in recent years has
focused on investigating Zta reactivation of latency and its role in lytic viral DNA
replication. This has revealed a wealth of knowledge about Zta as a
multifunctional transcription factor. However, a complete understanding of Zta
transcriptional activation is still missing. Here, utilising ChIP-qPCR, we showed
conserved binding patterns for Zta across several EBV lytic gene promoters in
two different EBV systems including a non-B cell EBV-infected cell line. Also,
using luciferase reporter assays, we show the first functional evidence for a
possible role of Zta in controlling transcription regulation at distal regulatory
elements (enhancers). Importantly, we identified BNLF2a, an essential viral
immune evasion gene, as a direct target for Zta. We identified five ZREs and
mapped functional ones within the BNLF2a promoter using mutational analysis
and luciferase reporter assays. We also expressed and purified a recombinant
GFP-bZIP Zta protein to address Zta binding to BNLF2a ZREs in vitro.
Interestingly, using in silico approach, we also identified a conserved sequence
in the ZRE flanking region of all five ZREs within BNLF2a promoter and
uncovered a role for a possible repressor at ZRE2 flanking region. Our work
not only adds to our understanding of Zta transcription regulation but
characterises for the first time the regulation of a novel Zta target that has a role
in evading the host immune system during EBV pre-latency and lytic cycle
A novel RNA Pol II CTD interaction site on the mRNA Capping Enzyme is essential for its allosteric activation
The RNA cap methyltransferases RNMT and CMTR1 co-ordinate gene expression during neural differentiation
Regulation of RNA cap formation has potent impacts on gene regulation, controlling which transcripts are expressed, processed and translated into protein. Recently, the RNA cap methyltransferases RNA guanine-7 methyltransferase (RNMT) and cap-specific mRNA (nucleoside-2'-O-)-methyltransferase 1 (CMTR1) have been found to be independently regulated during embryonic stem (ES) cell differentiation controlling the expression of overlapping and distinct protein families. During neural differentiation, RNMT is repressed and CMTR1 is up-regulated. RNMT promotes expression of the pluripotency-associated gene products; repression of the RNMT complex (RNMT–RAM) is required for repression of these RNAs and proteins during differentiation. The predominant RNA targets of CMTR1 encode the histones and ribosomal proteins (RPs). CMTR1 up-regulation is required to maintain the expression of histones and RPs during differentiation and to maintain DNA replication, RNA translation and cell proliferation. Thus the co-ordinate regulation of RNMT and CMTR1 is required for different aspects of ES cell differentiation. In this review, we discuss the mechanisms by which RNMT and CMTR1 are independently regulated during ES cell differentiation and explore how this influences the co-ordinated gene regulation required of emerging cell lineages
CMTR1 is recruited to transcription start sites and promotes ribosomal protein and histone gene expression in embryonic stem cells
CMTR1 (cap methyltransferase 1) catalyses methylation of the first transcribed nucleotide of RNAPII transcripts (N1 2′-O-Me), creating part of the mammalian RNA cap structure. In addition to marking RNA as self, N1 2′-O-Me has ill-defined roles in RNA expression and translation. Here, we investigated the gene specificity of CMTR1 and its impact on RNA expression in embryonic stem cells. Using chromatin immunoprecipitation, CMTR1 was found to bind to transcription start sites (TSS) correlating with RNAPII levels, predominantly binding at histone genes and ribosomal protein (RP) genes. Repression of CMTR1 expression resulted in repression of RNAPII binding at the TSS and repression of RNA expression, particularly of histone and RP genes. In correlation with regulation of histones and RP genes, CMTR1 repression resulted in repression of translation and induction of DNA replication stress and damage. Indicating a direct role for CMTR1 in transcription, addition of recombinant CMTR1 to purified nuclei increased transcription of the histone and RP genes. CMTR1 was found to be upregulated during neural differentiation and there was an enhanced requirement for CMTR1 for gene expression and proliferation during this process. We highlight the distinct roles of the cap methyltransferases RNMT and CMTR1 in target gene expression and differentiation
Mechanism of activation of the BNLF2a immune evasion gene of Epstein-Barr virus by Zta
The human gamma herpes virus Epstein–Barr virus (EBV) exploits multiple routes to evade the cellular immune response. During the EBV lytic replication cycle, viral proteins are expressed that provide excellent targets for recognition by cytotoxic T cells. This is countered by the viral BNLF2a gene. In B cells during latency, where BNLF2a is not expressed, we show that its regulatory region is embedded in repressive chromatin. The expression of BNLF2a mirrors the expression of a viral lytic cycle transcriptional regulator, Zta (BZLF1, EB1, ZEBRA), in B cells and we propose that Zta plays a role in up-regulating BNLF2a. In cells undergoing EBV lytic replication, we identified two distinct regions of interaction of Zta with the chromatin-associated BNLF2a promoter. We identify five potential Zta-response elements (ZREs) in the promoter that are highly conserved between virus isolates. Zta binds to these elements in vitro and activates the expression of the BNLF2a promoter in both epithelial and B cells. We also found redundancy amongst the ZREs. The EBV genome undergoes a biphasic DNA methylation cycle during its infection cycle. One of the ZREs contains an integral CpG motif. We show that this can be DNA methylated during EBV latency and that both Zta binding and promoter activation are enhanced by its methylation. In summary, we find that the BNLF2a promoter is directly targeted by Zta and that DNA methylation within the proximal ZRE aids activation. The implications for regulation of this key viral gene during the reactivation of EBV from latency are discussed