Transcriptional Function Research Articles

Mutant androgen receptor induces neurite loss and senescence independently of ARE binding in a neuronal model of SBMA

Spinal and bulbar muscular atrophy (SBMA) is a slowly progressing neuromuscular disease caused by a polyglutamine (polyQ)-encoding CAG trinucleotide repeat expansion in the androgen receptor (AR) gene, leading to AR aggregation, lower motor neuron death, and muscle atrophy. AR is a ligand-activated transcription factor that regulates neuronal architecture and promotes axon regeneration; however, whether AR transcriptional functions contribute to disease pathogenesis is not fully understood. Using a differentiated PC12 cell model of SBMA, we identified dysfunction of polyQ-expanded AR in its regulation of neurite growth and maintenance. Specifically, we found that in the presence of androgens, polyQ-expanded AR inhibited neurite outgrowth, induced neurite retraction, and inhibited neurite regrowth. This dysfunction was independent of polyQ-expanded AR transcriptional activity at androgen response elements (ARE). We further showed that the formation of polyQ-expanded AR intranuclear inclusions promoted neurite retraction, which coincided with reduced expression of the neuronal differentiation marker β-III-Tubulin. Finally, we revealed that cell death is not the primary outcome for cells undergoing neurite retraction; rather, these cells become senescent. Our findings reveal that mechanisms independent of AR canonical transcriptional activity underly neurite defects in a cell model of SBMA and identify senescence as a pathway implicated in this pathology. These findings suggest that in the absence of a role for AR canonical transcriptional activity in the SBMA pathologies described here, the development of SBMA therapeutics that preserve this activity may be desirable. This approach may be broadly applicable to other polyglutamine diseases such as Huntington's disease and spinocerebellar ataxias.

SHAPEwarp-web: sequence-agnostic search for structurally homologous RNA regions across databases of chemical probing data.

RNA molecules perform a variety of functions in cells, many of which rely on their secondary and tertiary structures. Chemical probing methods coupled with high-throughput sequencing have significantly accelerated the mapping of RNA structures, and increasingly large datasets of transcriptome-wide RNA chemical probing data are becoming available. Analogously to what has been done for decades in the protein world, this RNA structural information can be leveraged to aid the discovery of structural similarity to a known RNA (or RNA family), which, in turn, can inform about the function of transcripts. We have previously developed SHAPEwarp, a sequence-agnostic method for the search of structurally homologous RNA segments in a database of reactivity profiles derived from chemical probing experiments. In its original implementation, however, SHAPEwarp required substantial computational resources, even for moderately sized databases, as well as significant Linux command line know-how. To address these limitations, we introduce here SHAPEwarp-web, a user-friendly web interface to rapidly query large databases of RNA chemical probing data for structurally similar RNAs. Aside from featuring a completely rewritten core, which speeds up by orders of magnitude the search inside large databases, the web server hosts several high-quality chemical probing databases across multiple species. SHAPEwarp-web is available fromhttps://shapewarp.incarnatolab.com.

Detecting haplotype-specific transcript variation in long reads with FLAIR2

BackgroundRNA-seq has brought forth significant discoveries regarding aberrations in RNA processing, implicating these RNA variants in a variety of diseases. Aberrant splicing and single nucleotide variants (SNVs) in RNA have been demonstrated to alter transcript stability, localization, and function. In particular, the upregulation of ADAR, an enzyme that mediates adenosine-to-inosine editing, has been previously linked to an increase in the invasiveness of lung adenocarcinoma cells and associated with splicing regulation. Despite the functional importance of studying splicing and SNVs, the use of short-read RNA-seq has limited the community’s ability to interrogate both forms of RNA variation simultaneously.ResultsWe employ long-read sequencing technology to obtain full-length transcript sequences, elucidating cis-effects of variants on splicing changes at a single molecule level. We develop a computational workflow that augments FLAIR, a tool that calls isoform models expressed in long-read data, to integrate RNA variant calls with the associated isoforms that bear them. We generate nanopore data with high sequence accuracy from H1975 lung adenocarcinoma cells with and without knockdown of ADAR. We apply our workflow to identify key inosine isoform associations to help clarify the prominence of ADAR in tumorigenesis.ConclusionsUltimately, we find that a long-read approach provides valuable insight toward characterizing the relationship between RNA variants and splicing patterns.

Cellular and circuit architecture of the lateral septum for reward processing

The lateral septum (LS) is composed of heterogeneous cell types that are important for various motivated behaviors. However, the transcriptional profiles, spatial arrangement, function, and connectivity of these cell types have not been systematically studied. Using single-nucleus RNA sequencing, we delineated diverse genetically defined cell types in the LS that play distinct roles in reward processing. Notably, we found that estrogen receptor 1 (Esr1)-expressing neurons in the ventral LS (LSEsr1) are key drivers of reward seeking via projections to the ventral tegmental area, and these neurons play an essential role in methamphetamine (METH) reward and METH-seeking behavior. Extended exposure to METH increases the excitability of LSEsr1 neurons by upregulating hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, thereby contributing to METH-induced locomotor sensitization. These insights not only elucidate the intricate molecular, circuit, and functional architecture of the septal region in reward processing but also reveal a neural pathway critical for METH reward and behavioral sensitization.

Mechanomemory of nucleoplasm and RNA polymerase II after chromatin stretching by a microinjected magnetic nanoparticle force

Increasing evidence suggests that the mechanics of chromatin and nucleoplasm regulate gene transcription and nuclear function. However, how the chromatin and nucleoplasm sense and respond to forces remains elusive. Here, we employed a strategy of applying forces directly to the chromatin of a cell via a microinjected 200-nm anti-H2B-antibody-coated ferromagnetic nanoparticle (FMNP) and an anti-immunoglobulin G (IgG)-antibody-coated or an uncoated FMNP. The chromatin behaved as a viscoelastic gel-like structure and the nucleoplasm was a softer viscoelastic structure at loading frequencies of 0.1-5Hz. Protein diffusivity of the chromatin, nucleoplasm, and RNA polymerase II (RNA Pol II) and RNA Pol II activity were upregulated in a chromatin-stretching-dependent manner and stayed upregulated for tens of minutes after force cessation. Chromatin stiffness increased, but the mechanomemory duration of chromatin diffusivity decreased, with substrate stiffness. These findings may provide a mechanomemory mechanism of transcription upregulation and have implications on cell and nuclear functions.

Functional analysis of protein interactions using coupled bi-fluorescence complementation/GFP nanobody techniques.

Transcription factors (TFs) form homo- or hetero-dimeric DNA binding complexes along with associated co-regulators that can have transcriptional repressor or activator functions. Defining the specific composition of the complexes is therefore key to understanding their biological role. Here, we utilized bimolecular fluorescence complementation (BiFC) to visualize the formation of defined TF dimers and associated co-regulators derived from the activator protein-1 (AP-1) and myocyte enhancer factor 2 (MEF2) families. Firstly, BiFC signals were observed in cells co-expressing TFs tagged with complimentary combinations of the split fluorescent protein, demonstrating the engineered formation ofdefined dimer complexes. Next, we applied this approach and determined that defined AP-1 dimers localized at discrete sub-nuclear locations. Subsequently, a combination of BiFC coupled with GFP binding peptide (GBP)-nanotrap allowed observation of protein-protein interactions between a co-regulator, HDAC4, and defined BiFC-MEF2 engineered dimers. To determine transactivation properties of defined TF dimers in a cellular system, the Gal4-DNA binding domain fused to GBP was utilized to assess the transcriptional properties of the BiFC-TF dimers using a generically applicable Gal4/UAS luciferase reporter gene assay system. Here, we report efficacy of a BiFC/GBP-nanobody approach that allows engineering, visualization, and functional analysis of defined TF dimers.

WRKY Transcription Factor Responses and Tolerance to Abiotic Stresses in Plants.

Plants are subjected to abiotic stresses throughout their developmental period. Abiotic stresses include drought, salt, heat, cold, heavy metals, nutritional elements, and oxidative stresses. Improving plant responses to various environmental stresses is critical for plant survival and perpetuation. WRKY transcription factors have special structures (WRKY structural domains), which enable the WRKY transcription factors to have different transcriptional regulatory functions. WRKY transcription factors can not only regulate abiotic stress responses and plant growth and development by regulating phytohormone signalling pathways but also promote or suppress the expression of downstream genes by binding to the W-box [TGACCA/TGACCT] in the promoters of their target genes. In addition, WRKY transcription factors not only interact with other families of transcription factors to regulate plant defence responses to abiotic stresses but also self-regulate by recognising and binding to W-boxes in their own target genes to regulate their defence responses to abiotic stresses. However, in recent years, research reviews on the regulatory roles of WRKY transcription factors in higher plants have been scarce and shallow. In this review, we focus on the structure and classification of WRKY transcription factors, as well as the identification of their downstream target genes and molecular mechanisms involved in the response to abiotic stresses, which can improve the tolerance ability of plants under abiotic stress, and we also look forward to their future research directions, with a view of providing theoretical support for the genetic improvement of crop abiotic stress tolerance.

A BRD4‐Targeting Photothermal Agent for Controlled Protein Degradation

AbstractBRD4 protein plays a pivotal role in cell cycle regulation and differentiation. Disrupting the activity of BRD4 has emerged as a promising strategy for inhibiting the growth and proliferation of cancer cells. Herein, we introduced a BRD4‐targeting photothermal agent for controlled protein degradation, aiming to enhance low‐temperature photothermal therapy (PTT) for cancer treatment. By incorporating a BRD4 protein inhibitor into a cyanine dye scaffold, the photothermal agent specifically bond to the bromodomain of BRD4. Upon low power density laser irradiation, the agent induced protein degradation, directly destroying the BRD4 structure and inhibiting its transcriptional regulatory function. This strategy not only prolonged the retention time of the photothermal agent in cancer cells but also confined the targeted protein degradation process solely to the tumor tissue, minimizing side effects on normal tissues through the aid of exogenous signals. This work established a simple and feasible platform for future PTT agent design in clinical cancer treatment.

A BRD4-Targeting Photothermal Agent for Controlled Protein Degradation.

BRD4 protein plays a pivotal role in cell cycle regulation and differentiation. Disrupting the activity of BRD4 has emerged as a promising strategy for inhibiting the growth and proliferation of cancer cells. Herein, we introduced a BRD4-targeting photothermal agent for controlled protein degradation, aiming to enhance low-temperature photothermal therapy (PTT) for cancer treatment. By incorporating a BRD4 protein inhibitor into a cyanine dye scaffold, the photothermal agent specifically bond to the bromodomain of BRD4. Upon low power density laser irradiation, the agent induced protein degradation, directly destroying the BRD4 structure and inhibiting its transcriptional regulatory function. This strategy not only prolonged the retention time of the photothermal agent in cancer cells but also confined the targeted protein degradation process solely to the tumor tissue, minimizing side effects on normal tissues through the aid of exogenous signals. This work established a simple and feasible platform for future PTT agent design in clinical cancer treatment.

RUNX1 regulates MCM2/CDC20 to promote COAD progression modified by deubiquitination of USP31

Colon adenocarcinoma (COAD) is the second leading cause of cancer death, and there is still a lack of diagnostic biomarkers and therapeutic targets. In this study, bioinformatics analysis of the TCGA database was used to obtain RUNX1, a gene with prognostic value in COAD. RUNX1 plays an important role in many malignancies, and its molecular regulatory mechanisms in COAD remain to be fully understood. To explore the physiological role of RUNX1, we performed functional analyses, such as CCK-8, colony formation and migration assays. In addition, we investigated the underlying mechanisms using transcriptome sequencing and chromatin immunoprecipitation assays. RUNX1 is highly expressed in COAD patients and significantly correlates with survival. Silencing of RUNX1 significantly slowed down the proliferation and migratory capacity of COAD cells. Furthermore, we demonstrate that CDC20 and MCM2 may be target genes of RUNX1, and that RUNX1 may be physically linked to the deubiquitinating enzyme USP31, which mediates the upregulation of RUNX1 protein to promote transcriptional function. Our results may provide new insights into the mechanism of action of RUNX1 in COAD and reveal potential therapeutic targets for this disease.

Epitranscriptomic cytidine methylation of the hepatitis B viral RNA is essential for viral reverse transcription and particle production

Epitranscriptomic RNA modifications have emerged as important regulators of the fate and function of viral RNAs. One prominent modification, the cytidine methylation 5-methylcytidine (m5C), is found on the RNA of HIV-1, where m5C enhances the translation of HIV-1 RNA. However, whether m5C functionally enhances the RNA of other pathogenic viruses remains elusive. Here, we surveyed a panel of commonly found RNA modifications on the RNA of hepatitis B virus (HBV) and found that HBV RNA is enriched with m5C as well as ten other modifications, at stoichiometries much higher than host messenger RNA (mRNA). Intriguingly, m5C is mostly found on the epsilon hairpin, an RNA element required for viral RNA encapsidation and reverse transcription, with these m5C mainly deposited by the cellular methyltransferase NSUN2. Loss of m5C from HBV RNA due to NSUN2 depletion resulted in a partial decrease in viral core protein (HBc) production, accompanied by a near-complete loss of the reverse transcribed viral DNA. Similarly, mutations introduced to remove the methylated cytidines resulted in a loss of HBc production and reverse transcription. Furthermore, pharmacological disruption of m5C deposition led to a significant decrease in HBV replication. Thus, our data indicate m5C methylations as a critical mediator of the epsilon elements' function in HBV virion production and reverse transcription, suggesting the therapeutic potential of targeting the m5C methyltransfer process on HBV epsilon as an antiviral strategy.

Multiomic profiling of transcription factor binding and function in human brain.

Transcription factors (TFs) orchestrate gene expression programs crucial for brain function, but we lack detailed information about TF binding in human brain tissue. We generated a multiomic resource (ChIP-seq, ATAC-seq, RNA-seq, DNA methylation) on bulk tissues and sorted nuclei from several postmortem brain regions, including binding maps for more than 100 TFs. We demonstrate improved measurements of TF activity, including motif recognition and gene expression modeling, upon identification and removal of high TF occupancy regions. Further, predictive TF binding models demonstrate a bias for these high-occupancy sites. Neuronal TFs SATB2 and TBR1 bind unique regions depleted for such sites and promote neuronal gene expression. Binding sites for TFs, including TBR1 and PKNOX1, are enriched for risk variants associated with neuropsychiatric disorders, predominantly in neurons. This work, titled BrainTF, is a powerful resource for future studies seeking to understand the roles of specific TFs in regulating gene expression in the human brain.

Expression characterization of miR‐6826‐3p in Pardosa pseudoannulata (Araneae: Lycosidae) under high temperature stress

AbstractPardosa pseudoannulata is key predator in rice ecosystems; it plays a crucial role in rice pest biological control, effectively managing major rice pests such as rice planthopper. At present, there are few related studies on spiders' microRNA (miRNA). Consequently, we initiate a study on miR‐6826‐3p in P. pseudoannulata in response to high temperatures. First, we identified a total of 686 target genes of miR‐6826‐3p, 439 target genes had annotated results in the database, primarily involved in transcription and metabolism functions. Subsequently, quantitative real‐time PCR was employed to assess the relative expression levels of miR‐6826‐3p in P. pseudoannulata under different temperatures (31, 34, 37, 40°C) and various durations (1, 2, 4, 8, 12 h). The results revealed that compared to the control group (25°C), the relative expression level of miR‐6826‐3p in female spiders significantly increased under the same temperature and different duration stresses. The expression level of miR‐6826‐3p in male spiders showed an initial increase followed by a decrease trend, with relatively lower expression levels after 1 and 12 h at the same temperature conditions. Furthermore, under the same duration of stress, compared to the control group (25°C), the relative expression level of miR‐6826‐3p in female spiders significantly increased at different temperatures. These results demonstrate that P. pseudoannulata miR‐6826‐3p plays a significant role in response to high‐temperature stress. The research findings can provide a basis for future in‐depth studies on the miRNA functional of P. pseudoannulata, aiding in the elucidation of the regulatory mechanisms of P. pseudoannulata miRNAs in response to high‐temperature environments.

A Comparative Full-Length Transcriptome Analysis Using Oxford Nanopore Technologies (ONT) in Four Tissues of Bovine Origin.

The transcriptome complexity and splicing patterns in male and female cattle are ambiguous, presenting a substantial obstacle to genomic selection programs that seek to improve productivity, disease resistance, and reproduction in cattle. A comparative transcriptomic analysis using Oxford Nanopore Technologies (ONT) was conducted in bovine testes (TESTs), ovaries (OVAs), muscles (MUSCs), and livers (LIVs). An average of 5,144,769 full-length reads were obtained from each sample. The TESTs were found to have the greatest number of alternative polyadenylation (APA) events involved in processes such as sperm flagellum development and fertilization in male reproduction. In total, 438 differentially expressed transcripts (DETs) were identified in the LIVs in a comparison of females vs. males, and 214 DETs were identified in the MUSCs between females and males. Additionally, 14,735, 36,347, and 33,885 DETs were detected in MUSC vs. LIV, MUSC vs. TEST, and OVA vs. TEST comparisons, respectively, revealing the complexity of the TEST. Gene Set Enrichment Analysis (GSEA) showed that these DETs were mainly involved in the "spermatogenesis", "flagellated sperm motility", "spermatid development", "reproduction", "reproductive process", and "microtubule-based movement" KEGG pathways. Additional studies are necessary to further characterize the transcriptome in different cell types, developmental stages, and physiological conditions in bovines and ascertain the functions of the novel transcripts.

MsBERT-Promoter: a multi-scale ensemble predictor based on BERT pre-trained model for the two-stage prediction of DNA promoters and their strengths

BackgroundA promoter is a specific sequence in DNA that has transcriptional regulatory functions, playing a role in initiating gene expression. Identifying promoters and their strengths can provide valuable information related to human diseases. In recent years, computational methods have gained prominence as an effective means for identifying promoter, offering a more efficient alternative to labor-intensive biological approaches.ResultsIn this study, a two-stage integrated predictor called “msBERT-Promoter” is proposed for identifying promoters and predicting their strengths. The model incorporates multi-scale sequence information through a tokenization strategy and fine-tunes the DNABERT model. Soft voting is then used to fuse the multi-scale information, effectively addressing the issue of insufficient DNA sequence information extraction in traditional models. To the best of our knowledge, this is the first time an integrated approach has been used in the DNABERT model for promoter identification and strength prediction. Our model achieves accuracy rates of 96.2% for promoter identification and 79.8% for promoter strength prediction, significantly outperforming existing methods. Furthermore, through attention mechanism analysis, we demonstrate that our model can effectively combine local and global sequence information, enhancing its interpretability.ConclusionsmsBERT-Promoter provides an effective tool that successfully captures sequence-related attributes of DNA promoters and can accurately identify promoters and predict their strengths. This work paves a new path for the application of artificial intelligence in traditional biology.

CBX4 plays a bidirectional role in transcriptional regulation and lung adenocarcinoma progression

Lung adenocarcinoma (LUAD) remains a leading cause of cancer-related mortality worldwide. Understanding the dysregulated epigenetics governing LUAD progression is pivotal for identifying therapeutic targets. CBX4, a chromobox protein, is reported to be upregulated in LUAD. This study highlights the dual impact of CBX4 on LUAD proliferation and metastasis through a series of rigorous in vitro and in vivo experiments. Further investigation into the underlying mechanism through high-throughput ChIP-seq and RNA-seq reveals that CBX4 functions in promoting LUAD proliferation via upregulating PHGDH expression and subsequent serine biosynthesis, while concurrently suppressing LUAD metastasis by inhibiting ZEB2 transcription. CBX4 facilitates PHGDH transcription through the interaction with GCN5, inducing heightened histone acetylation on the PHGDH promoter. Simultaneously, the inhibition of ZEB2 transcription involves CBX4-mediated recruitment of canonical PRC1 (cPRC1), establishing H2K119ub on the ZEB2 promoter. These findings underscore CBX4’s pivotal role as a regulator of LUAD progression, emphasizing its diverse transcriptional regulatory functions contingent upon interactions with specific epigenetic partners. Understanding the nuanced interplay between CBX4 and epigenetic factors sheds light on potential therapeutic avenues in LUAD.

DNA-binding protein-A promotes kidney ischemia/reperfusion injury and participates in mitochondrial function

DNA-binding protein-A (DbpA; gene: Ybx3) belongs to the cold shock protein family with known functions in cell cycling, transcription, translation, and tight junction communication. In chronic nephritis, DbpA is upregulated. However, its activities in acute injury models, such as kidney ischemia/reperfusion injury (IRI), are unclear. To study this, mice harboring Ybx3+/+, Ybx3+/- or the Ybx3-/- genotype were characterized over 24 months and following experimental kidney IRI. Mitochondrial function, number and integrity were analyzed by mitochondrial stress tests, MitoTracker staining and electron microscopy. Western Blot, immunohistochemistry and flow cytometry were performed to quantify tubular cell damage and immune cell infiltration. DbpA was found to be dispensable for kidney development and tissue homeostasis under healthy conditions. Furthermore, endogenous DbpA protein localizes within mitochondria in primary tubular epithelial cells. Genetic deletion of Ybx3 elevates the mitochondrial membrane potential, lipid uptake and metabolism, oxygen consumption rates and glycolytic activities of tubular epithelial cells. Ybx3-/- mice demonstrated protection from IRI with less immune cell infiltration, endoplasmic reticulum stress and tubular cell damage. A presumed protective mechanism was identified via upregulated antioxidant activities and reduced ferroptosis, when Ybx3 was deleted. Thus, our studies reveal DbpA acts as a mitochondrial protein with profound adverse effects on cell metabolism and highlights a protective effect against IRI when Ybx3 is genetically deleted. Hence, preemptive DbpA targeting in situations with expected IRI, such as kidney transplantation or cardiac surgery, may preserve post-procedure kidney function.

A novel noncanonical function for IRF6 in the recycling of E-cadherin.

Interferon Regulatory Factor 6 (IRF6) is a transcription factor essential for keratinocyte cell-cell adhesions. Previously, we found that recycling of E-cadherin was defective in the absence of IRF6, yet total E-cadherin levels were not altered, suggesting a previously unknown, nontranscriptional function for IRF6. IRF6 protein contains a DNA binding domain (DBD) and a protein binding domain (PBD). The transcriptional function of IRF6 depends on its DBD and PBD, however, whether the PBD is necessary for the interaction with cytoplasmic proteins has yet to be demonstrated. Here, we show that an intact PBD is required for recruitment of cell-cell adhesion proteins at the plasma membrane, including the recycling of E-cadherin. Colocalizations and coimmunoprecipitations reveal that IRF6 forms a complex in recycling endosomes with Rab11, Myosin Vb, and E-cadherin, and that the PBD is required for this interaction. These data indicate that IRF6 is a novel effector of the endosomal recycling of E-cadherin and demonstrate a non-transcriptional function for IRF6 in regulating cell-cell adhesions.

M6A reader YTHDF1 promotes cardiac fibrosis by enhancing AXL translation.

Cardiac fibrosis caused by ventricular remodeling and dysfunction such as post-myocardial infarction (MI) can lead to heart failure. RNA N6-methyladenosine (m6A) methylation has been shown to play a pivotal role in the occurrence and development of many illnesses. In investigating the biological function of the m6A reader YTHDF1 in cardiac fibrosis, adeno-associated virus 9 was used to knock down or overexpress the YTHDF1 gene in mouse hearts, and MI surgery in vivo and transforming growth factor-β (TGF-β)-activated cardiac fibroblasts in vitro were performed to establish fibrosis models. Our results demonstrated that silencing YTHDF1 in mouse hearts can significantly restore impaired cardiac function and attenuate myocardial fibrosis, whereas YTHDF1 overexpression could further enhance cardiac dysfunction and aggravate the occurrence of ventricular pathological remodeling and fibrotic development. Mechanistically, zinc finger BED-type containing 6 mediated the transcriptional function of the YTHDF1 gene promoter. YTHDF1 augmented AXL translation and activated the TGF-β-Smad2/3 signaling pathway, thereby aggravating the occurrence and development of cardiac dysfunction and myocardial fibrosis. Consistently, our data indicated that YTHDF1 was involved in activation, proliferation, and migration to participate in cardiac fibrosis in vitro. Our results revealed that YTHDF1 could serve as a potential therapeutic target for myocardial fibrosis.

Genome-Wide Transcriptional Roles of KSHV Viral Interferon Regulatory Factors in Oral Epithelial Cells.

The viral interferon regulatory factors (vIRFs) of KSHV are known to dysregulate cell signaling pathways to promote viral oncogenesis and to block antiviral immune responses to facilitate infection. However, it remains unknown to what extent each vIRF plays a role in gene regulation. To address this, we performed a comparative analysis of the protein structures and gene regulation of the four vIRFs. Our structure prediction analysis revealed that despite their low amino acid sequence similarity, vIRFs exhibit high structural homology in both their DNA-binding domain (DBD) and IRF association domain. However, despite this shared structural homology, we demonstrate that each vIRF regulates a distinct set of KSHV gene promoters and human genes in epithelial cells. We also found that the DBD of vIRF1 is essential in regulating the expression of its target genes. We propose that the structurally similar vIRFs evolved to possess specialized transcriptional functions to regulate specific genes.